Methods and Compositions for Sequentially Detecting Targets

ABSTRACT

Compositions, kits and methods for detecting a plurality of targets are provided herein. A probe-set composition is provided, including one or more first probes and one or more second probes. Each of the first probe includes a nucleic acid sequence complementary to a nucleic acid barcode of a corresponding target-specific binding partner, a first label, and a cleavage site for a first cleavage agent, wherein the first cleavage agent is capable of releasing the first label. Each of the second probes includes a nucleic acid sequence complementary to a nucleic acid barcode of a corresponding target-specific binding partner, a second label, a quench moiety that renders the second label undetectable, and a cleavage site for the first cleavage agent. The first cleavage agent is capable of releasing the quench moiety, whereby the second label is rendered detectable.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication No. 62/780,038, filed Dec. 14, 2018, the content of which isincorporated herein by reference in its entirety for any purpose.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicformat. The Sequence Listing is provided as a file entitled“2019-12-11_01168-0019-00US_SEQ_List_ST25.txt” created on Dec. 11, 2019,which is 8,192 bytes in size. The information in the electronic formatof the sequence listing is incorporated herein by reference in itsentirety.

INTRODUCTION

This application relates generally to the field of detection of analytes(e.g., targets), and in particular, relates to compositions, kits andmethods for detecting a plurality of targets.

In recent years, methods for examining biologically relevant molecules(known as biomarkers) in tissue samples have improved. The ability todetect increasing numbers of biomarkers has permitted more sophisticatedcharacterization of tissue architecture that, in turn, allows cliniciansto better understand and predict an individual's health condition byexamining a tissue sample. Whereas previous methods involved examiningone biomarker at a time, multiplex immunohistochemistry (IHC) allowsspatial profiling of multiple biomarkers in a single tissue sample. IHCinvolves binding antibodies to biomarkers, attaching fluorescent labelsto the antibodies (with each biomarker identified by a different colorfluorescent label) and using a fluorescence microscope to image thesample.

DNA Exchange Imaging is an established process for multiplexed detectionof biomarkers in tissue samples, which employs multiple rounds oftreatment of the tissue. First, DNA-barcoded antibodies are applied tothe sample and allowed to bind to corresponding targets. Next, an“imager strand” is applied to the sample; this binds to the DNA-barcodeof one of the targets. A coverslip is placed on the sample, and theimager strand is then detected using a fluorescence microscope. Next,the coverslip is removed; the sample is treated to remove the imagerstrand, and a second imager strand is applied to the sample; this bindsto the second DNA-barcode. The second imager strand is then detected.The coverslip is again removed, and the sample treated to remove thesecond imager strand in preparation for round three. This process may berepeated to detect increasing numbers of biomarkers.

Thus, DNA Exchange Imaging requires application of new imager strandsbefore each round of detection. Applying a collection of imager strandsthat would function effectively through multiple rounds of detectionwould reduce the amount of manipulation (and thus, time) required tocomplete an analysis. Such manipulation would be further reduced ifcoverslip removal could be avoided.

SUMMARY

In accordance with the description, methods and compositions forsequentially detecting targets in a sample are provided.

Accordingly, the following embodiments according to the methods andcompositions described herein are provided.

Embodiment 1. A method for detecting a plurality of target molecules,the method comprising:

-   (a) contacting a sample with two or more target-specific binding    partners, wherein each target-specific binding partner comprises a    nucleic acid barcode; and is specific for a different target    molecule;-   (b) contacting the sample with one or more probe-sets wherein each    probe in a probe-set is specific for a different target-specific    binding partner, and wherein each probe-set comprises: a first    probe, comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a first label; and    -   a cleavage site for a first cleavage agent, wherein the first        cleavage agent is capable of releasing the first label, and    -   a second probe comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a second label;    -   a quench moiety, wherein the quench moiety renders the second        label undetectable; and    -   a cleavage site for the first cleavage agent, wherein the first        cleavage agent is capable of releasing the quench moiety,        whereby the second label is rendered detectable; and optionally        comprises a cleavage site for a second cleavage agent wherein        the second cleavage agent is capable of releasing the second        label;-   (c) detecting signals corresponding to labels of the first probes of    each of the one or more probe-sets;-   (d) contacting the sample with a first cleavage agent, thereby    releasing the labels of the first probes in each of the one or more    probe-sets; and releasing the quench moieties of the second probes    in each of the one or more probe-sets, thereby activating signals    corresponding to the second labels, and-   (e) detecting signals corresponding to the labels of the second    probes of each of the one or more probe-sets.

Embodiment 2. The method of Embodiment 1, wherein one or more of theprobe-sets further comprises a third probe, and the method furthercomprises:

-   (f) in step (b), contacting the sample with the third probe    comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a third label; and    -   a quench moiety, wherein the quench moiety renders the third        label undetectable; and    -   a cleavage site for the second cleavage agent, wherein the        second cleavage agent is capable of releasing the quench moiety,        whereby the third label is rendered detectable;    -   and optionally comprises a distinct cleavage site for a distinct        cleavage agent capable of releasing the third label; and-   (g) after step (e), contacting the sample with a second cleavage    agent, thereby releasing the labels of the second probes in each of    the one or more probe-sets; and releasing the quench moieties of the    third probes in one or more probe-sets, thereby activating signals    corresponding to the third labels; and-   (h) detecting signals corresponding to the labels of the third    probes of one or more probe sets.

Embodiment 3. The method of Embodiment 2, wherein one or more of theprobe-sets further comprise a subsequent probe, and the method furthercomprises:

-   (i) in step (b), contacting the sample with a subsequent probe    contained in one or more probe-set, wherein the subsequent probe    comprises:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a subsequent label; and    -   a quench moiety, wherein the quench moiety renders the        subsequent label undetectable; and    -   a cleavage site for a subsequent cleavage agent, wherein the        subsequent cleavage agent is capable of releasing the quench        moiety, whereby the subsequent label is rendered detectable;    -   and optionally comprises a distinct cleavage site for a distinct        cleavage agent capable of releasing activated labels from probes        in the sample;-   (j) contacting the sample with a subsequent cleavage agent, thereby    releasing activated labels of the probes in each probe-set; and    releasing the quench moieties of the subsequent probes in each    probe-set, thereby activating signals corresponding to the labels of    the subsequent probes in each probe-set; and-   (k) detecting signals corresponding to the labels of the subsequent    probes of each probe-set; and-   (l) optionally repeating steps (i) through (k).

Embodiment 4. The method of Embodiment 1, wherein the first and seconddetectable labels of a probe-set are the same.

Embodiment 5. The method of Embodiment 1, wherein the first and seconddetectable labels of a probe-set are different.

Embodiment 6. The method of Embodiment 2, wherein the two or more of thefirst, second, and third detectable labels of a probe-set are the same.

Embodiment 7. The method of Embodiment 2, wherein the two or more of thefirst, second, and third detectable labels of a probe-set are different.

Embodiment 8. The method of Embodiment 3, wherein two or more of thefirst, second, third, and subsequent labels are the same.

Embodiment 9. The method of Embodiment 3, wherein two or more of thefirst, second, third, and subsequent labels are different.

Embodiment 10. The method of Embodiment 1, further comprising washingthe sample after contacting the sample with the first cleavage agentand/or after contacting the sample with the second cleavage agent.

Embodiment 11. The method of Embodiment 1, wherein the sample is notwashed after contacting the sample with the first cleavage agent and/orafter contacting the sample with the second cleavage agent.

Embodiment 12. The method of Embodiment 11, wherein the coverslip is notremoved.

Embodiment 13. The method of Embodiment 1, further comprising increasingthe number of nucleic acid barcodes on a target-specific bindingpartner, wherein multiple copies of a corresponding probe bind tomultiple copies of the nucleic acid barcode.

Embodiment 14. The method of Embodiment 13, wherein the number ofnucleic acid bar codes is increased using rolling circle amplification,primer exchange reaction, hybridization chain reaction, or DNAbranching.

Embodiment 15. The method of Embodiment 14, wherein the number ofnucleic acid bar codes is increased before the target-specific bindingpartner is contacted with the sample.

Embodiment 16. The method of Embodiment 14, wherein the number ofnucleic acid bar codes is increased when the target-specific bindingpartner is bound to its target molecule.

Embodiment 17. The method of Embodiment 1, wherein the released label ofa first probe comprises a nucleotide sequence.

Embodiment 18. The method of Embodiment 17, further comprisingcontacting the sample with a background-reducing agent comprising anucleotide sequence complementary to that of the released label of thereleased first probe, wherein binding of the background-reducing agentto the released label of the released first probe quenches the signal ofthe label.

Embodiment 19. The method of Embodiment 2, wherein the released label ofa second probe comprises a nucleotide sequence.

Embodiment 20. The method of Embodiment 19, further comprisingcontacting the sample with a background-reducing agent comprising anucleotide sequence complementary to that of the released label of thesecond probe, wherein binding of the background-reducing agent to thelabel of the released second probe quenches the signal of the label.

Embodiment 21. The method of Embodiment 3, wherein an activated label ofa released probe comprises a nucleotide sequence.

Embodiment 22. The method of Embodiment 16, further comprisingcontacting the sample with a background-reducing agent comprising anucleotide sequence complementary to that of the released label of theprobe, wherein binding of the background-reducing agent to the releasedlabel of the released probe quenches the signal of the label.

Embodiment 23. A probe-set composition comprising:

-   one or more first probes, each comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a first label; and    -   a cleavage site for a first cleavage agent, wherein the first        cleavage agent is capable of releasing the first label, and-   one or more second probes, each comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a second label;    -   a quench moiety, wherein the quench moiety renders the second        label undetectable; and    -   a cleavage site for the first cleavage agent, wherein the first        cleavage agent is capable of releasing the quench moiety,        whereby the second label is rendered detectable;    -   and optionally comprises a distinct cleavage site for a distinct        cleavage agent capable of releasing the second label.

Embodiment 24. The composition of Embodiment 23, further comprising:

-   one or more third probes, each comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a third label;    -   a quench moiety, wherein the quench moiety renders the third        label undetectable; and    -   a cleavage site for a second cleavage agent, wherein the second        cleavage agent is capable of releasing the quench moiety,        whereby the third label is rendered detectable;    -   and optionally comprises a distinct cleavage site for a distinct        cleavage agent capable of releasing the third label,-   wherein the second probe further comprises a cleavage site for the    second cleavage agent.

Embodiment 25. The composition of Embodiment 24, further comprising:

-   a subsequent probe comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a subsequent label;    -   a quench moiety for the subsequent label, wherein the quench        moiety renders the subsequent label undetectable; and    -   a cleavage site, wherein a cleavage agent is capable of        releasing the quench moiety, whereby the subsequent label is        rendered detectable;    -   and optionally comprises a distinct cleavage site wherein a        distinct cleavage agent is capable of releasing the subsequent        label,-   wherein another probe in the probe-set comprises a cleavage site for    the same cleavage agent that releases the quench moiety of the    subsequent probe.

Embodiment 26. The composition of Embodiment 23, wherein the one or morefirst probes have the same label.

Embodiment 27. The composition of Embodiment 23, wherein the one or morefirst probes have a different label.

Embodiment 28. The composition of Embodiment 23, wherein the cleavagesite is an electromagnetic cleavage site; a chemical cleavage site; or amechanical cleavage site.

Embodiment 29. The composition of Embodiment 28, wherein theelectromagnetic cleavage site is a photocleavage site.

Embodiment 30. The composition of Embodiment 29, wherein thephotocleavage site is an ultraviolet (UV) cleavage site.

Embodiment 31. The composition of Embodiment 23, wherein the first orsecond label is a fluorescent label.

Embodiment 32. The composition of Embodiment 23, further comprising: abackground reducing agent comprising a nucleotide sequence complementaryto a nucleotide sequence of a portion of a first probe present betweenthe cleavage site and the first label.

Embodiment 33. The composition of Embodiment 32, further comprising: abackground reducing agent comprising a nucleotide sequence complementaryto a nucleotide sequence of a portion of a second probe present betweenthe cleavage site and the second label.

Embodiment 34. The composition of Embodiment 33, further comprising: abackground reducing agent comprising a nucleotide sequence complementaryto a nucleotide sequence of a portion of a subsequent probe presentbetween the cleavage site and the subsequent label.

Embodiment 35. A kit, comprising:

-   the probe-set composition of Embodiment 23;-   a background-reducing agent;-   a coverslip;-   one or more target-specific binding partners;-   one or more buffers;-   one or more reagents for increasing the number of nucleic acid    barcodes of a target-specific binding partner;-   one or more cleavage agents;-   a nuclear counterstain; and-   instructions for use.

Embodiment 36. The kit of Embodiment 35, wherein the background-reducingagent is linked to a solid phase.

Embodiment 37. The kit of Embodiment 36, wherein the solid phase isselected from a coverslip; a particle and a slide.

Embodiment 38. The kit of Embodiment 35, wherein the background-reducingagent is in liquid phase.

Embodiment 39. A background reducing agent, comprising a nucleotidesequence complementary to a released label of a first probe of thecomposition of Embodiment 23, and a quench material.

Embodiment 40. A background reducing agent, comprising a nucleotidesequence complementary to a released label of a second probe of thecomposition of Embodiment 24, and a quench material.

Embodiment 41. A background reducing agent, comprising a nucleotidesequence complementary to a released activated label of a probe of thecomposition of Embodiment 25, and a quench material.

Embodiment 42. A method for detecting a plurality of target molecules,the method comprising:

-   (a) contacting a sample with two or more target-specific binding    partners, wherein each target-specific binding partner comprises a    nucleic acid barcode; and is specific for a different target    molecule;-   (b) contacting the sample with one or more probe-sets wherein each    probe in a probe-set is specific for a different target-specific    binding partner, and wherein each probe-set comprises:    -   a first probe, comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a first label; and    -   a cleavage site for a first cleavage agent, wherein the first        cleavage agent is capable of suppressing the first label, and-   a second probe comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a second label;    -   a quench moiety, wherein the quench moiety renders the second        label undetectable; and    -   a cleavage site for the first cleavage agent, wherein the first        cleavage agent is capable of releasing or suppressing the quench        moiety, whereby the second label is rendered detectable;    -   and optionally comprises a cleavage site wherein a second        cleavage agent is capable of releasing or suppressing the second        label;-   (c) detecting signals corresponding to labels of the first probes of    each of the one or more probe-sets;-   (d) contacting the sample with a first cleavage agent, thereby    suppressing the labels of the first probes in each of the one or    more probe-sets; and releasing or suppressing the quench moieties of    the second probes in each of the one or more probe-sets, thereby    activating signals corresponding to the second labels; and-   (e) detecting signals corresponding to the labels of the second    probes of each of the one or more probe-sets.

Embodiment 43. The method of Embodiment 42, wherein one or more of theprobe-sets further comprises a third probe, comprising:

-   (f) in step (b), contacting the sample with a third probe contained    in one or more probe-sets, wherein the third probe comprises:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a third label;    -   a quench moiety, wherein the quench moiety renders the third        label undetectable; and    -   a cleavage site for a second cleavage agent, wherein the second        cleavage agent is capable of releasing or suppressing the quench        moiety, whereby the third label is rendered detectable;    -   and optionally comprises a distinct cleavage site wherein a        distinct cleavage agent is capable of releasing or suppressing        the third label of one or more probe-sets; and-   (g) after step (e), contacting the sample with a second cleavage    agent, thereby suppressing the labels of the second probes in each    of the one or more probe-sets; and releasing or suppressing the    quench moieties of the third probes in one or more probe-sets,    thereby activating signals corresponding to the third labels; and-   (h) detecting signals corresponding to the third labels.

Embodiment 44. The method of Embodiment 43, wherein one or more of theprobe-sets further comprise a subsequent probe, comprising:

-   (i) in step (b), contacting the sample with a subsequent probe    contained in one or more probe-set, wherein the subsequent probe    comprises:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a subsequent label; and    -   a quench moiety, wherein the quench moiety renders the        subsequent label undetectable; and    -   a cleavage site, wherein a subsequent cleavage agent is capable        of releasing or suppressing the quench moiety, whereby the        subsequent label is rendered detectable;    -   and optionally comprises a distinct cleavage site wherein a        distinct cleavage agent is capable of suppressing activated        labels from probes in the sample;-   (j) contacting the sample with a subsequent cleavage agent, thereby    suppressing activated labels of the probes in each probe-set; and    releasing the quench moieties of the subsequent probes in each    probe-set, thereby activating signals corresponding to the    subsequent labels; and-   (k) detecting signals corresponding to the subsequent labels; and-   (l) optionally repeating steps (i) through (k).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D schematically depicts an exemplary probe-set of the presentdisclosure, containing a first probe (FIG. 1A); second probe (FIG. 1B),third probe (FIG. 1C); and fourth probe (FIG. 1D).

FIGS. 2A-E provide schematics of embodiments disclosed herein. FIG. 2Aschematically depicts three exemplary probe-sets (probe-sets A, B, andC) bound to nine target-specific binding partners (depicted asantibody-DNA conjugates). Probe-set A is directed to targets 1, 4 and 7.Probe-set B is directed to targets 2, 5 and 8. Probe-set C is directedto targets 3, 6 and 9. The exemplary probe-sets are designed for threerounds of detection: Round 1—targets 1, 2, and 3; Round 2—targets 4, 5and 6; and Round 3,—targets 7, 8 and 9.

FIG. 2B schematically depicts the probe-sets bound to a prepared tissuesample fixed to a microscope slide, in accordance with an embodiment ofthe present disclosure. FIGS. 2C, 2D, and 2E schematically depict first,second, and third rounds of detection, respectively.

FIGS. 3A-C schematically depict an exemplary embodiment of a methoddescribed herein, where first, second and third probes of the probe-setdepicted in FIG. 1 are bound to target-specific binding partners havingmultiple nucleic acid barcode repeats to which cognate probes bind(FIGS. 3A, 3B, and 3C, respectively).

FIGS. 4A-D schematically depict four exemplary cleavage modes useful invarious embodiments of the present disclosure. FIG. 4A depicts use ofultraviolet (UV) light as a cleavage agent for a photocleavable bond.FIG. 4B depicts use of Tris(2-carboxyethyl)phosphine (TCEP) as acleavage agent for a disulfide bond. FIG. 4C depicts use of arestriction enzyme as a cleavage agent for a specific nucleotidesequence. FIG. 4D depicts use of uracil-DNA glycosylase as a cleavageagent for a nucleic acid hairpin containing uracil-glycosidic bonds.

FIGS. 5A-D schematically depict an exemplary embodiment of the presentdisclosure in which three targets are detected after application ofthree probes of a probe-set, using three rounds of detection; and thennew probes are applied to the sample and subsequent rounds of detectionare performed.

FIGS. 6A-B show fluorescence microscope images of a tissue sampleprocessed according to an embodiment of the present disclosure, after afirst round of detection of four targets (6A) and second round ofdetection of four different targets (6B).

FIGS. 7A-B show images of a first round of detection in which the probeis quenched (7A) and second round of detection in which the probe isunquenched upon treatment with a cleavage agent (7B), in a tissue sampleusing an embodiment of the present disclosure.

FIGS. 8A-D shows segments of exemplary probes of the present disclosure.

FIG. 9 schematically depicts an exemplary non-selectivebackground-reducing agent.

FIG. 10 schematically depicts exemplary embodiments of selectivebackground-reducing agents.

FIGS. 11A-C show schematic depictions of designs of abackground-reducing agent and images from rounds of detection using thebackground-reducing agent. FIG. 11A schematically depicts embodiments ofdesigns of a background-reducing agent, a corresponding first probe(Probe design A) and a second probe (Probe design B). FIGS. 11B-C showimages from two rounds of detection using the designs of FIG. 11A: afirst round (FIG. 11B) in which CD8, PD1, PDL1 and CD68 were detectedsimultaneously; and a second round (FIG. 11C) in which CD3, CD4, FoxP3and Cytokeratin were detected simultaneously, in accordance with thepresent disclosure.

FIGS. 12A-D shows images of detecting a target in tissue. The figuresshow images without coverslip removal, in the absence (FIGS. 12A/12B))or presence (FIGS. 12C/12D)) of a background-reducing agent.

FIGS. 13A-B show schematics of exemplary probe sets and images generatedfrom reactions using those probe sets. Specifically, FIG. 13Aschematically depicts an exemplary probe-set containing four members,designed for four rounds of detection using three different cleavageagents (TCEP, UV photocleavage; and uracil-DNA-glycosylase). FIG. 13Bshows images of sixteen detected target molecules using four probe-setsaccording to the probe-set embodiment shown in FIG. 13A.

DESCRIPTION

Described herein are probe-sets for multiplex detection of targetmolecules (or “targets”), related methods and kits for using them, andrelated background-reducing agents. In an embodiment, the methods can beused to detect multiple target molecules with a single up-frontapplication of target-binding partners and labeled probes, followed bysequential detection of subpopulations of probes. In variousembodiments, a multiplex method can be implemented without direct accessto the sample after initial application of probes, for example, withoutremoving a coverslip enclosing a tissue sample.

Although embodiments of the invention are explained in detail, it is tobe understood that other embodiments are contemplated. Accordingly, itis not intended that the invention is limited in its scope to thedetails of construction and arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or carried out invarious ways. Also, in describing the embodiments, specific terminologywill be resorted to for the sake of clarity.

It must also be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,reference to a sheet or portion is intended also to include themanufacturing of a plurality of sheets or portions. References to asheet containing “a” constituent is intended to include otherconstituents in addition to the one named.

Also, in describing the embodiments, terminology will be resorted to forthe sake of clarity. It is intended that each term contemplates itsbroadest meaning as understood by those skilled in the art and includesall technical equivalents which operate in a similar manner toaccomplish a similar purpose.

Ranges may be expressed herein as from “about” or “approximately” oneparticular value and/or to “about” or “approximately” another particularvalue. When such a range is expressed, another embodiment includes fromthe one particular value and/or to the other particular value.

By “comprising” or “containing” or “including” is meant that at leastthe named compound, element, particle, or method step is present in thecomposition or article or method, but does not exclude the presence ofother compounds, materials, particles, method steps, even if the othersuch compounds, material, particles, method steps have the same functionas what is named.

It is also to be understood that the mention of one or more method stepsdoes not preclude the presence of additional method steps or interveningmethod steps between those steps expressly identified. Similarly, it isalso to be understood that the mention of one or more components in afabric or system does not preclude the presence of additional componentsor intervening components between those components expressly identified.

Methods for multiplexed detection of targets in tissue samples usingfluorescence microscopy have been described, for example, in U.S. Pat.No. 9,944,972 and are commercially available. The InSituPlex multiplexdetection technology (Ultivue, Inc.) is used for detecting multipletargets as follows. First, a collection of DNA-barcoded antibodies forfour targets are applied to the sample, where the DNA-barcodes aredistinct for each antibody. After amplification of the DNA-barcodes,fluorescently labeled probes that bind to the barcodes are applied. Thefluorescent labels are spectrally distinct and all can be imagedsimultaneously. Additional targets may be examined by removing theprobes, and applying another collection of probes complementary to asecond set of barcoded antibodies. Thus, in an assay designed to examinenine targets using this method, the user could image the first threetargets then perform exchange by removing the probes and applying thenext round of three probes. Targets four through six would then beimaged, and exchange would again be performed. Targets seven throughnine would then be imaged.

In contrast, the methods described herein employ probe-sets that can besimultaneously applied to a sample, and sequentially detected withoutthe need for an exchange step. FIGS. 1A-D (depicting Probes A, B, C, andD, respectively) schematically depict an exemplary probe-set of thepresent disclosure. Although a variety of variations are possible, thisfigure shows a basic implementation of the present disclosure. In thisembodiment, each probe (A, B, C, and D) is specific for a differenttarget, and each contains the same label. Probes B, C, and D contain thesame quench moiety, which is capable of quenching the label. Probes Aand B contain a common cleavage site (C₁), which permits simultaneousrelease of the label of Probe A, and de-quenching of the label of ProbeB. Probes B and C contain a common cleavage site (C₂), which likewisepermits simultaneous release of the label of Probe B, and de-quenchingof the label of Probe C. Probes C and D contain a common cleavage site(C₃), which permits simultaneous release of the label of Probe C, andde-quenching of the label of Probe D. Probe D optionally contains acleavage site (C₄), which permits release of the label of Probe D, andoptionally, de-quenching of the label of a subsequent probe.

FIG. 2A schematically depicts three exemplary probe-sets bound totarget-specific binding partners (TSBPs) in accordance with the presentdisclosure. In this embodiment, nine different targets are detectedusing three rounds of detection, with members of probe-sets A, B, and Cused in each round. TSBPs are depicted as antibodies having nucleic acidsegments, each having a distinct barcode to which a distinct probebinds. The probes of each probe-set are depicted as bound to theirmatching TSBPs. The probes within a particular probe-set contain acommon label (see FIG. 2A legend). Each different probe-set has adifferent label and corresponding quench moiety in this embodiment. FIG.2B schematically depicts the exemplary probe-bound TSBPs of FIG. 2A whenbound to targets of a tissue sample on a slide.

FIG. 2C schematically depicts Round 1 of detection, where in thisembodiment, the labels of the first probes (targets 1, 2 and 3) of eachprobe-set are detected. The detectable probes are depicted as blackprobe/TSBP/target complexes; the non-detectable probes are depicted ingrey probe/TSBP/target complexes.

FIG. 2D schematically depicts Round 2 of detection, which is preceded byapplication of a cleavage agent that releases the labels of the firstprobes of each probe-set and releases the quench moiety of the secondprobes of each probe-set. Release of the quench moieties renders thelabels of the second probes of each probe-set detectable, and release ofthe labels of the first probes removes their signals from theTSBP/target complexes. Accordingly, the now-detectable second probes(targets 4, 5 and 6) of each probe-set are depicted as blackprobe/TSBP/target complexes.

FIG. 2E schematically depicts Round 3 of detection, which is preceded byapplication of a second cleavage agent that releases the labels of thesecond probes of each probe-set and releases the quench moiety of thethird probes of each probe-set. Accordingly, the now-detectable thirdprobes (targets 7, 8 and 9) of each probe-set are depicted as blackprobe/TSPB/target complexes. A label cleavage site may be present orabsent from a probe used in a final round of detection because a usermay or may not desire to remove the last-detected signal.

FIGS. 3A-3C schematically depict an exemplary embodiment of a methoddescribed herein, where first, second and third probes of the probe-setare bound to TSBP, where each TSBP contains multiple identical nucleicacid barcode repeats to which cognate probes bind. Binding of multipleprobes to each TSBP is an exemplary method for amplifying the detectablesignals of the probes.

FIGS. 4A-4D schematically depict four exemplary cleavage modes useful invarious embodiments of the present disclosure. In FIG. 4A, the quenchedlabel of a probe bound to a TSPB/target is activated when UV light isused as a cleavage agent for a photocleavable bond. In FIG. 4B, thequenched label is activated when Tris(2-carboxyethyl)phosphine (TCEP) isused as a cleavage agent for a disulfide bond. In FIG. 4C, the quenchedlabel is activated when a restriction enzyme is used as a cleavage agentfor a specific nucleotide sequence. In FIG. 4D, the quenched label isactivated when uracil-DNA glycosylase is used as a cleavage agent thathydrolyzes uracil-glycosidic (UA) bonds, destabilizing a DNA duplex.

FIGS. 5A-5B schematically depict an exemplary embodiment of thedescribed methods, which illustrates that serial probe-sets may be usedto detect increasing numbers of targets. The illustration depicts that afirst target is detected in round A (FIG. 5A); the sample is treatedwith TCEP to release the first label and unquench the second label; asecond target is detected in round B (FIG. 5B); the sample is thentreated with UV light to release the second label and unquench the thirdlabel; a third target is detected in round C (FIG. 5C); the sample isnext treated with uracil DNA glycosylase to release the third label;another probe-set is added to the sample, and a fourth target isdetected in round D (FIG. 5D); the user continues with one or morerounds of detection using any selected cleavage modes to detectadditional targets. Other types of probes also may be used with theprobe-set described herein to detect additional targets.

Therefore, the present disclosure provides compositions, kits andmethods for detecting a plurality of targets. Provided herein is aprobe-set composition. The composition includes one or more firstprobes, each containing a nucleic acid sequence complementary to anucleic acid barcode of a corresponding target-specific binding partner;a first label; and a cleavage site for a first cleavage agent, whereinthe first cleavage agent is capable of releasing the first label, andone or more second probes, each containing nucleic acid sequencecomplementary to a nucleic acid barcode of a correspondingtarget-specific binding partner; a second label; a quench moiety,wherein the quench moiety renders the second label undetectable; and acleavage site for the first cleavage agent, wherein the first cleavageagent is capable of releasing the quench moiety, whereby the secondlabel is rendered detectable; and optionally comprises a distinctcleavage site for a distinct cleavage agent capable of releasing thesecond label.

In some embodiments, a probe-set can further include one or more thirdprobes, each comprising: a nucleic acid sequence complementary to anucleic acid barcode of a corresponding target-specific binding partner;a third label; a quench moiety, wherein the quench moiety renders thethird label undetectable; and a cleavage site for a second cleavageagent, wherein the second cleavage agent is capable of releasing thequench moiety, whereby the third label is rendered detectable; andoptionally comprises a distinct cleavage site for a distinct cleavageagent capable of releasing the third label, wherein the second probefurther comprises a cleavage site for the second cleavage agent.

In some embodiments, a probe-set can be expanded to include a subsequentprobe comprising: a nucleic acid sequence complementary to a nucleicacid barcode of a corresponding target-specific binding partner; asubsequent label; a quench moiety for the subsequent label, wherein thequench moiety renders the subsequent label undetectable; and a cleavagesite, wherein a cleavage agent is capable of releasing the quenchmoiety, whereby the subsequent label is rendered detectable; andoptionally comprises a distinct cleavage site wherein a distinctcleavage agent is capable of releasing the subsequent label, whereinanother probe in the probe-set comprises a cleavage site for the samecleavage agent that releases the quench moiety of the subsequent probe.

The term “first probe” means a probe that contains at least (1) anucleic acid sequence complementary to a nucleic acid barcode of acorresponding target-specific binding partner; (2) a first label; and(3) a cleavage site for a first cleavage agent, wherein the firstcleavage agent is capable of releasing the first label. A first probecan further contain one or more additional quench moieties, one or moreother labels, and one or more other cleavage sites that are deployed orundeployed in a method step. Elements that remain undeployed in aparticular method (e.g., a photocleavage site is present when nophotocleavage agent is employed in the method) can be used inalternative methods, giving the user flexibility when designing assayflows. A probe may have more than one cleavage site for the samecleavage agent, or distinct cleavage sites for distinct cleavage agentsthat do not interfere with subsequent steps. Such redundancies may beuseful, for example, for weaker cleavage agents, to create shorterreleased probe fragments, or other purposes. Similarly, a second, third,and subsequent probe can contain any number of quench moieties, labels,and/or cleavage sites. Therefore, in some embodiments, some probes of aprobe-set may contain one or more labels, quench moieties, or cleavagesites that are not deployed. In some embodiments, the cleavage productof a probe may contain more than one label, quench moiety, and/orundeployed cleavage site. It is foreseeable that cleavage of a probe canrelease more than one cleavage product. If desired, a first probe cancontain a quench moiety such that a cleavage agent is contacted with asample prior to detecting the label of a first probe.

If desired, two or more probes can be designed to produce a proximitysignal that reflects the physical distance between two or more targetsusing fluorescence resonance energy transfer (FRET). In an embodiment ofthis implementation, a probe contains a label (e.g., fluorophore) thatis capable of being quenched by a label (e.g., quencher) contained onanother probe, when the probes are bound to targets in close proximity.Therefore, a FRET signal of a donor or acceptor label can be detected inone or more rounds of detection as described herein.

FIGS. 8A-8D depict segments of exemplary probes encompassed by thepresent disclosure. FIGS. 8A-8C show exemplary first probe segmentscontaining a disulfide cleavage site and a terminal label. The number ofnucleotides, or physical distance imparted by other chemistry, can varyas selected by the user. In FIG. 8B, ‘T’ is a thymine base. FIG. 8Dshows an exemplary second probe segment containing a disulfide cleavagesite between a label and a quench moiety.

It may be desired to reduce the signal from labels released from probesprior to detecting the labels of the next-to-be-detected probes. Abackground-reducing agent can be employed for this purpose. As usedherein, the term “background-reducing agent” means a material that bindsto a released label of a probe and reduces the label's signal, whichotherwise could produce unwanted or background signal. Abackground-reducing agent can reduce signal by one or more mechanisms,including direct or indirect quenching of the label; relocation of thelabel outside of a selected imaging field; or both. Abackground-reducing agent can bind non-selectively or selectively to areleased label.

In an embodiment, a non-selective background-reducing agent is a carbonnanoparticle carbon nanomaterial including macroscopic, mesoscopic, andnanoscale materials, including single crystal carbon, carbon sheets,glassy carbon, carbon black, carbon pastes, activated carbon, graphene,carbon nanotubes, graphene oxide, and carbon dots. Exemplary size rangesfor particles include one dimension less than 20 nm. Graphene oxideparticles possess hydroxyl and carboxyl groups in addition to grapheniccarbon. While the carboxyl groups impart ionicity that favors solubilityin aqueous solutions, the high degree of charge on the particlespromotes surface adsorption as well. Thus, useful carbon nanoparticlesmay exhibit a high degree of hydroxyls relative to carboxyls. Grapheneoxide has been used, for example, to reduce unwanted fluorescence intissue microscopy (see, for example, Li, R., Georgiades, P., Cox, H. etal. Quenched Stochastic Optical Reconstruction Microscopy (qSTORM) withGraphene Oxide. Sci Rep 8, 16928 (2018) doi:10.1038/s41598-018-35297-4).As depicted in FIG. 9, a released label can bind to graphene oxide,which can be in solution or attached to a solid support (e.g. acoverslip, slide or particle), and subsequently signal from the releasedlabel is suppressed or quenched.

In an embodiment, a selective background-reducing agent bindsspecifically to a nucleotide sequence contained in a released label. Inan embodiment, such a selective background-reducing agent binds to thereleased label, resulting in quenching of the label when a quencher ofthe background-reducing agent is brought into proximity with the label.This is useful, for example, when performing a cleavage without removalof the coverslip, such as the UV photocleavage embodiments describedherein.

Therefore, in an embodiment, a nucleic acid selectivebackground-reducing agent is a molecule or complex comprising (i) anucleotide sequence complementary to a nucleotide sequence contained ina probe segment comprising a label, which is released from a probe uponcleavage by a cleavage agent; (ii) a quencher capable of quenching thesignal of the label; and optionally (iii) a cleavage site whereby thebackground-reducing agent is itself activated or made available forinteracting with the probe segment (e.g., in the case of a cagedbackground-reducing agent) or released from a solid support (e.g., inthe case of release from a coverslip, microscope slide, bead and thelike).

In some embodiments, the background-reducing agent is linked to a solidsupport, such as a sample enclosure (e.g., a coverslip, slide,capillary) or accessory (e.g., bead, particle). When a solid support isused, the background-reducing agent can remain associated with thesupport and thereby attract released labels to the support.Alternatively, the background-reducing agent can be liberated from thesupport by a cleavage agent, which can be the same as the cleavage agentused to release its partner released label, or a different cleavageagent. FIG. 10 shows background-reducing agent initially attached to asupport and subsequently cleaved from the support and bound to releasedlabels (e.g., FIG. 10, a); and background-reducing agent that remainsattached to a support when bound to released labels (e.g., FIG. 10, b).

A background-reducing agent can have a variety of structures. In anembodiment, a background-reducing agent comprises a circularized nucleicacid. A circularized nucleic acid may include a cleavage site wherebycleavage renders the background-reducing agent available for interactingwith its partner released label (e.g., FIG. 10, c). In an embodiment, abackground-reducing agent comprises a nucleic acid hairpin structure(e.g., FIG. 10, d). In some embodiments, a background-reducing agentcomprises a nucleic acid hairpin structure comprising one or morecleavage sites whereby cleavage renders the background-reducing agentavailable for interacting with its partner released label (e.g., FIG.10, e, f, g).

In an embodiment, a background-reducing agent comprises a cleavage sitewhereby cleavage renders the agent activated or available forinteracting with its partner released label. Cleavage sites and cleavageagents are described herein below; any of a variety of such cleavagesites and cleavage agents are suitable for use in a background-reducingagent. In some embodiments, a background-reducing agent comprising cagedmolecules that block interaction with partner released labels, until acleavage agent is used to uncage the blockers, allowing the agent tointeract with its partner released label (e.g., FIG. 10, h).

The interaction between a background-reducing agent and its partnerreleased label can be non-covalent or covalent. Exemplary non-covalentinteractions include binding between complementary or partiallycomplementary nucleic acids. Exemplary covalent interactions includecross-linking between complementary or partially complementary nucleicacids (e.g., FIG. 10, i; 3-cyanovinylcarbazole nucleosidephotocrosslinker (CNVK)). Thus, a background-reducing agent may becapable of cross-linking to a released label. This can be useful whenusing a relatively lower affinity binding site for a released label,such as a nucleic acid sequence that binds transiently under the assayconditions employed.

Any cleavage agent described herein can be used for abackground-reducing agent. In a specific embodiment, abackground-reducing agent can be activated by a photocleavage agent. Asdescribed in Example 7 below, when a photocleavage agent is used in afluorescence microscopy method, the sample can be treated with aphotocleavage wavelength of light (e.g., 385 nm) using the imagingsystem without removal from the stage used for acquiring a first (orsubsequent) round of images. Alternatively, the sample can be removedfrom the imaging system; externally treated with the photocleavageagent; and returned to the imaging system (or another detection mode)for a next detection round. As described elsewhere in this disclosure,images from different rounds can be aligned using well known methods.

A composition containing a background-reducing agent can be provided inany form, such as dried, solubilized in a compatible liquid, in acolloidal mixture, and together with other components, for example, forconvenience of a particular workflow. As such, a background-reducingagent can be a component of a background-reducing mounting medium.

A background-reducing mounting medium is a fluid to be disposed betweena tissue and a coverslip, which includes one or more quenchers selectivefor one or more labels. The fluid can contain one or more of, or acombination of: salts, buffers, hardening agents, anti-fade agents, andstaining reagents (e.g., a nuclear counterstain). Examples of commercialmounting reagents that can be used as a base for adding abackground-reducing agent include Prolong Gold Anti-Fade Mountant(ThermoFisher); and Vectashield (Vecta Laboratories). Examples of commonreagents that can be used as a base for adding affinity and/or quenchmaterials include a variety of buffers used for biological samples suchas phosphate buffered saline (PBS) and tris(hydroxymethyl)aminomethane(Tris)-based buffers.

The present disclosure provides kits for multiplex detection of aplurality of target molecules. In an embodiment, a kit includes one ormore probe-sets as described herein together with one or more othercomponents. Each probe-set includes one or more first probes and one ormore second probes. Optionally one or more third probes; fourth probes,fifth probes; sixth probes, or more probes can be included. The one ormore other kit components can include one or more of instructions foruse; one or more background-reducing agents; one or more cleavageagents; one or more target-specific binding partners; one or morebuffers; one or more reagents for increasing the number of nucleic acidbarcodes of a target-specific binding partner; a nuclear counterstain; abackground-reducing mounting medium; a coverslip; plate; control sample;software; and other component.

The present disclosure provides methods for detecting a plurality oftarget molecules. In an embodiment, the method involves: (a) contactinga sample with two or more target-specific binding partners, wherein eachtarget-specific binding partner comprises a nucleic acid barcode; and isspecific for a different target molecule; (b) contacting the sample withone or more probe-sets wherein each probe in a probe-set is specific fora different target-specific binding partner, and wherein each probe-setcomprises a first probe, comprising a nucleic acid sequencecomplementary to a nucleic acid barcode of a correspondingtarget-specific binding partner; a first label; and a cleavage site fora first cleavage agent, wherein the first cleavage agent is capable ofreleasing the first label, and a second probe comprising a nucleic acidsequence complementary to a nucleic acid barcode of a correspondingtarget-specific binding partner; a second label; a quench moiety,wherein the quench moiety renders the second label undetectable; and acleavage site for a first cleavage agent, wherein the first cleavageagent is capable of releasing the quench moiety, whereby the secondlabel is rendered detectable; and optionally comprises a cleavage sitefor a second cleavage agent, wherein the second cleavage agent iscapable of releasing the second label; (c) detecting signalscorresponding to labels of the first probes of each probe-set; (d)contacting the sample with a first cleavage agent, thereby releasing thelabels of the first probes in each probe-set; and releasing the quenchmoieties of the second probes in each probe-set, thereby activatingsignals corresponding to the second labels of the second probes in eachprobe-set; and (e) detecting signals corresponding to the labels of thesecond probes of each probe-set.

In some embodiments, one or more of the probe-sets further comprises athird probe and the method described herein involves, at step (b),contacting the sample with the third probe containing a nucleic acidsequence complementary to a nucleic acid barcode of a correspondingtarget-specific binding partner; a third label; and a quench moiety,wherein the quench moiety renders the third label undetectable; and acleavage site for the second cleavage agent, wherein the second cleavageagent is capable of releasing the quench moiety, whereby the third labelis rendered detectable; and optionally comprises a distinct cleavagesite for a distinct cleavage agent capable of releasing the third labelof one or more probe-sets; and the method further comprises, after step(e), contacting the sample with a second cleavage agent, therebyreleasing the labels of the second probes in each of the one or moreprobe-sets; and releasing the quench moieties of the third probes in oneor more probe-sets, thereby activating signals corresponding to thelabels of the one or more third probes; and detecting signalscorresponding to the labels of the one or more third probes.

In an embodiment, the method described herein involves use of one ormore of the probe-sets containing a subsequent probe. The methodinvolves, (i) in step (b), contacting the sample with a subsequent probecontained in one or more probe-set, wherein the subsequent probecomprises: a nucleic acid sequence complementary to a nucleic acidbarcode of a corresponding target-specific binding partner; a subsequentlabel; and a quench moiety, wherein the quench moiety renders thesubsequent label undetectable; and a cleavage site for a subsequentcleavage agent, wherein a subsequent cleavage agent is capable ofreleasing the quench moiety, whereby the subsequent label is rendereddetectable; and optionally comprises a distinct cleavage site wherein adistinct cleavage agent is capable of releasing activated labels fromprobes in the sample; (j) contacting the sample with a subsequentcleavage agent, thereby releasing activated labels of the probes in eachprobe-set; and releasing the quench moieties of the subsequent probes ineach probe-set, thereby activating signals corresponding to the labelsof the subsequent probes in each probe-set; and (k) detecting signalscorresponding to the labels of the subsequent probes of each probe-set;and (1) optionally repeating steps (i) through (k).

The method can further include, in step (b), contacting the sample withtwo or more probe-sets, herein each probe-set further comprises asubsequent probe, comprising: a nucleic acid sequence complementary to anucleic acid barcode of a corresponding target-specific binding partner;a subsequent label; and a quench moiety, wherein the quench moietyrenders the subsequent label undetectable; and a cleavage site, whereina subsequent cleavage agent is capable of releasing the quench moiety,whereby the subsequent label is rendered detectable; and optionallycomprises a distinct cleavage site wherein a distinct cleavage agent iscapable of releasing the an activated label from probes in the sample;(j) after step (h), contacting the sample with a subsequent cleavageagent, thereby releasing activated labels of the probes in eachprobe-set; and releasing the quench moieties of the subsequent probes ineach probe-set, thereby activating signals corresponding to the labelsof the subsequent probes in each probe-set; and (k) detecting signalscorresponding to the labels of the subsequent probes of each probe-set;and (l) optionally repeating steps (i) through (k).

In various embodiments, the first and second detectable labels can bethe same; the first, second, and third detectable labels can be thesame; and the first, second, third and subsequent detectable labels canbe the same. Because the first, second, third, and subsequent detectablelabels in a probe-set are detected in first, second, third, andsubsequent rounds of detection, the labels need not be different (butcan be different). However, the first, second, third, and subsequentdetectable labels of one probe-set are different from those of adifferent probe-set, when detected in a first, second, third, andsubsequent round of detection.

When multiple probe-sets are used in a fluorescence detection mode, itcan be convenient to detect all first probes using the same detectionchannel. For example, when all labels are FITC-like, detection in asingle detection channel is possible. It is not necessary that allprobes contain the same detectable label for them to be detected in thesame detection channel, as a variety of fluorescent dyes are detectablein the same detection channel, as described in more detail below.Detection in more than one detection channel in a single round, as wellas more than one detection mode, can be used.

In various embodiments, the method can involve increasing the number ofnucleic acid barcodes contained in a TSBP, wherein multiple copies of acorresponding probe bind to multiple copies of the nucleic acid barcode.The number of barcodes can be increased using a method such as PCR,rolling circle amplification, primer exchange reaction (PER), HCR,branched amplification, or a combination of two or more methods. Themethod can be performed prior to addition of the target-specific bindingpartner to the sample, or after the target-specific binding partner iscontacted with the sample.

In some embodiments, the target may be a polypeptide or a nucleic acid.Accordingly, a target-specific binding partner can contain atarget-binding functionality that recognizes a polypeptide, nucleicacid, or other target.

In some embodiments, the cleavage site may be a chemical cleavage site;a mechanical cleavage site; an electromagnetic cleavage site, such as aphotocleavage site; or an enzymatic cleavage site.

In some embodiments, the label is a fluorescent label. As describedbelow, multiple fluorescent labels can be simultaneously selected usingroutine methods.

In various embodiments of the described methods, the sample can bewashed after contact with a cleavage agent. This is useful when thereleased label retains sufficient signal to produce unwanted backgroundsignal.

In some embodiments, the sample is not washed after contact with acleavage agent. This is useful when performing the method on a tissuesample covered by a coverslip, when it is desired to not remove thecoverslip.

In some embodiments the released label of a first probe comprises anucleotide sequence. In such embodiments, a method can further comprisecontacting the sample with a background-reducing agent comprising anucleotide sequence complementary to that of the released label of thereleased first probe, wherein binding of the background-reducing agentto the released label of the released first probe quenches the signal ofthe released first label.

Similarly, in some embodiments the released label of a second probecomprises a nucleotide sequence. In such embodiments, a method canfurther comprise contacting the sample with a background-reducing agentcomprising a nucleotide sequence complementary to that of the releasedlabel of the second probe, wherein binding of the background-reducingagent to the label of the released second probe quenches the signal ofthe released second label.

Likewise, in some embodiments, an activated label of a released probecomprises a nucleotide sequence. In such embodiments, a method canfurther comprise contacting the sample with a background-reducing agentcomprising a nucleotide sequence complementary to that of the releasedlabel of the probe, wherein binding of the background-reducing agent tothe released label of the released probe quenches the signal of thelabel.

The methods and compositions described herein can be used to detect avariety of types of targets. Exemplary types of targets includemacromolecules such as proteins, carbohydrates, lipids, and nucleicacids (e.g., DNA, RNA, short interfering nucleic acid (siNA), and shortinterfering RNA (siRNA)); and small molecules such as primarymetabolites, secondary metabolites, and natural products. A target canbe naturally occurring, in that it is present in organisms or virusesthat exist in nature in the absence of human intervention, or can besynthetic. The methods and compositions can be used to detect any targetfor which a target-specific binding partner exists.

Therefore, in some embodiments, a target is a protein. Examples ofprotein targets include cytoplasmic, nuclear, membrane, and non-cellularproteins, such as structural proteins (e.g. actin, vimentin, dystrophin,keratin); extracellular matrix proteins (e.g. elastin, fibronectin);cellular receptors (e.g. epidermal growth factor receptor, nerve growthfactor receptor, estrogen receptor); ion channels (e.g. GABA receptor,nicotinic acetylcholine receptor); hormones (e.g. insulin, oxytocin,androgens); DNA-binding proteins (e.g. P53, histones); immune systemproteins (e.g. CD3, CD4, CD8, CD20, CH11c, CD25, CD45RO, CD68, CD163,granzyme B, FoxP3, LAG3, MCHII, PD1, and PDL-1); and any other proteinof interest.

In some embodiments, a target is a nucleic acid molecule. Examples ofnucleic acid molecule targets include DNA and RNA. Example 5 belowdescribes an embodiment in which miRNA targets are detected.

As used herein, a “target-specific binding partner” means a molecule (orcomplex of molecules) that both (1) binds selectively to a target and(2) binds selectively to a probe. As such, a target-specific bindingpartner forms a molecular complex that includes both a target and aprobe. A target-specific binding partner has affinity for the targetsuch that it does not substantially bind to other molecules in thesample to the extent that such nonspecific binding interferes with thedesired experimental outcome, for example by creating unwantedbackground signal.

The target-binding functionality and the probe-binding functionality canbe contained within one molecule or two or more non-covalently boundmolecules. For instance, Example 1 below describes target-specificbinding partners that contain an antibody portion that providestarget-binding functionality, and a nucleic acid barcode portion thatprovides probe-binding functionality. A variety oftarget-specific-binding partners comprising antibody portions andnucleic acid portions are described, for example, in U.S. Pat. No.9,944,972.

The target-binding functionality of a target-specific binding partnercan be imparted, for example, by an antibody, an antibody fragment(e.g., Fab, Fab′, F(ab′)2, single heavy chain, diabody, and the like),an aptamer, a polypeptide, peptide (e.g., a ligand), a nucleic acid, orsmall molecule (e.g., a suicide substrate of an enzyme of interest). Thetarget-binding functionality will generally be selected based on thecharacter of the target. For example, when the target is a protein, anantibody can often provide the needed selective target-bindingfunctionality.

Similarly, the probe-binding functionality of a target-specific bindingpartner will be matched to the probe used. For example, when the probecontains a nucleic acid sequence, a complementary nucleic acid sequencecan provide the needed probe-biding functionality. However,probe-binding functionality can be imparted by a variety of partners,including by an antibody, an antibody fragment (e.g., Fab, Fab′,F(ab′)2, single heavy chain, diabody, and the like), an aptamer, apolypeptide, peptide (e.g., a ligand), a nucleic acid, small molecule(e.g., a suicide substrate of an enzyme of interest), and the like.

A nucleic acid portion of a target-specific binding partner, whether itimparts target-specific binding functionality or probe-specific bindingfunctionality, and a nucleic acid portion of a background-reducingagent, can contain DNA, RNA, a nucleic acid analog (for example, nucleicacid containing an altered phosphate backbone, an altered pentose sugar,and/or altered nucleobases, such as a nucleic acid containing2′-O-Methyl ribonucleic acid, 2′-fluoro ribonucleic acid, peptidenucleic acid, morpholino and locked nucleic acid, glycol nucleic acid,and threose nucleic acid), or a combination thereof. The nucleic acidportion can be double stranded, single stranded, or a combinationthereof.

As generally used herein, the term “nucleic acid” means a polymeric formof nucleotides of any length, such as deoxyribonucleotides orribonucleotides, or analogs thereof. For example, a nucleic acid may beDNA, RNA or the DNA product of RNA subjected to reverse transcription.Non-limiting examples of nucleic acids include coding or non-codingregions of a gene or gene fragment, loci (locus) defined from linkageanalysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomalRNA, ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. Other examples of nucleicacids include, without limitation, cDNA, aptamers, and peptide nucleicacids (“PNA”). A nucleic acid may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs (“analogous” forms ofpurines and pyrimidines are well known in the art). If present,modifications to the nucleotide structure may be imparted before orafter assembly of the polymer. A nucleic acid may be a single-stranded,double-stranded, partially single-stranded, or partially double-strandedDNA or RNA, depending on the application.

The term “nucleic acid barcode,” as used herein means a single strandednucleic acid segment, contained within a target-specific bindingpartner, that binds selectively to its complementary sequence within acognate probe. The number of nucleotides in a barcode will be sufficientto permit specific complementary binding to the probe under selectedreaction conditions. Those skilled in the art can design or empiricallydetermine barcode and matching complementary sequences suitable for useunder selected temperatures, salt concentrations, and other reactionconditions. Software resources for designing nucleic acid moleculesinclude, for example, www.nupack.org; Vienna RNA secondary structureserver (see, e.g., Vienna RNA secondary structure server Hofacker,Nucleic Acids Research, Volume 31, Issue 13, 1 July 2003, Pages3429-3431; Abdulkadir Elmas, Guido H. Jajamovich, Xiaodong Wang, andMichael S. Samoilov. Nucleic Acid Therapeutics, Volume: 23 Issue 2: Apr.4, 2013. Accordingly, a barcode can be, for example, between about 5 to20 nucleotides long, between about 8 to 15 nucleotides long, and betweenabout 10 to 14 nucleotides long.

Each nucleic acid barcode contains a known sequence, allowing eachtarget-specific binding partner used in a particular assay to bedistinctly identified by its barcode. FIG. 2A illustrates nine differenttarget-specific binding partners bound, via barcodes, to complementaryregions on probes. Each barcode is distinct, permitting nine distinctprobes to be used to identify each target-specific binding partner.Exemplary barcodes and corresponding complementary sequences are shownin Table 1, below.

TABLE 1 SEQ SEQ ID Complementary ID Barcode sequence NO sequence NO5′- ACGGAACCAACA -3′  1 5′- TGTTGGTTCCGT -3′ 13 5′- ACGGAATGAGGC -3′  25′- GCCTCATTCCGT -3′ 14 5′- ACTTGCTGACGA -3′  3 5′- TCGTCAGCAAGT -3′ 155′- TCACGTCAGCAT -3′  4 5′- ATGCTGACGTGA -3′ 16 5′- TTGACGATGGCA -3′  55′- TGCCATCGTCAA -3′ 17 5′- GGGAAGTAGGGC -3′  6 5′- GCCCTACTTVCC -3′ 185′- CCCAAAACGTCG -3′  7 5′- CGACGTTTTGGG -3′ 19 5′- TCGCTGTCATGA -3′  85′- TCATGACAGCGA -3′ 20 5′- AGCAATTCGGGT -3′  9 5′- ACCCGAATTGCT -3′ 215′- CGGGTTAAGGGT -3′ 10 5′- ACCCTTAACCCG -3′ 22 5′- GCGTTGGGATGA -3′ 115′- TCATCCCAACGC -3′ 23 5′- AGCGAGGAAAGT -3′ 12 5′- ACTTTCCTCGCT -3′ 24

A probe useful in the methods described herein can contain one or morenucleic acid barcodes, which can be present on the probe when contactedwith a sample, or generated by amplification when contacted with thesample. FIGS. 3A-3C depict a probe-set bound to target-specific bindingpartners having multiple barcodes. A variety of nucleic acidamplification methods can be used to increase the number of nucleic acidbarcodes contained in a target-specific binding partner. Exemplarymethods include polymerase chain reaction (PCR) (see, for example,McPherson M J, S G Moller, R Beynon, and C Howe 2000 PCR: Basics fromBackground to Bench. Heidelberg: Springer-Verlag); rolling circleamplification (RCA) (see, for example, Ali M M et al. Rolling circleamplification: A versatile tool for chemical biology, materials scienceand medicine. Chemical Society Reviews. 2014; 43(10):3324-3341); primerexchange reaction (PER) (see, for example, WO 2017/143006 A1; Kishi, etal. Nat Chem., 10(2): 155-164); DNA toehold-based strand displacement(see, for example, Schweller et al. PMCID: PMC3517005); hybridizationchain reaction (HCR) (see, for example, Dirks et al., 2014, PMID:15492210, 24712299); DNA hairpin-based dendrimerization reaction (see,for example, Yin et al., 2008, PMID 18202654); and any other method.Multiple types of amplification can even be used in combination. Forexample, Gusev et al reported combining rolling circle amplification andHRP-based signal amplification (Gusev, Y et al. Am. J. Pathology, vol.159,1 (2001): 63-9. doi:10.1016/S0002-9440(10)61674-4). Amplificationmethods have been described for target-specific binding partnerscontaining DNA barcodes, for example, in US 2018/0164308A1, which isincorporated herein by reference. Therefore, amplified nucleic acidbarcodes can be present in a variety of structural forms, includinglinear and branched forms, and can be within a target-specific bindingpartner or in one or more molecules bound to a target-specific bindingpartner, as described in US 2018/0164308A1.

In an embodiment, a nucleic acid barcode is amplified by RCA using acircular DNA template. The template is bound to the nucleic acidsequence of the target-specific binding partner; polymerase is added;and concatemeric repeats of the nucleic acid barcode are created. In anembodiment, a nucleic acid barcode is amplified by PER, whichisothermally produces single-stranded DNA with user-prescribed sequencesusing a strand-displacing polymerase. As described in Kishi, et al. NatChem., 10(2): 155-164, the PER process begins by designing a primer.Here, the primer would contain a nucleic acid barcode sequence orportion thereof. Utilizing a catalytic DNA hairpin mediator, PER thenappends to the existing primer a new primer with an independent,user-specified sequence. The newly extended primer can then trigger thenext step extension, thus forming a programmable PER cascade toautonomously grow a nascent DNA strand along a prescribed pathway toproduce a user-prescribed sequence.

A nucleic acid barcode of a target-specific binding partner can beamplified prior to contact with the sample or while in contact with thesample. When amplification is performed in contact with the sample,probes are generally applied after amplification. In some embodiments,the number of barcodes is greater than one; greater than two; greaterthan five; greater than 10; greater than 20; greater than 50; greaterthan 100.

Detection of Labels

A variety of detection modalities can be applied to the describedmethods. A label can be detected, for example, by an optical signal, anelectromagnetic signal (across the entire electromagnetic spectrum), anatomic/molecular mass (e.g., detectable by mass spectrometry), tangiblemass (e.g., detectable by atomic force microscope), an electrical signal(e.g., current or voltage); a mechanical signal (e.g., acoustic,pressure or other signal) and other methods. Specific detection methodsinclude light spectroscopy, fluorescence spectroscopy, RAMANspectroscopy, mass spectrometry, ion mobility spectrometry, secondaryion mass spectrometry (SIMS), Auger electron spectroscopy, X-rayphotoelectron spectroscopy (XPS), surface plasmon resonance, and myriadother detection methods. The type of detection selected will thereforedepend on the label(s) used. The type of sample and assay format willalso influence choice of detection mode. The examples below describe useof detection modes including fluorescence microscopy (which can produceimages in a direct or reconstructed manner); a fluorescence spectrometerin a plate reader; and a fluorescence spectrometer in a flow cytometrydevice. Any device or instrument capable of detecting a signal producedby a selected label can be used in a method described herein.

Labels

Given that a variety of detection modalities can be used with theprobe-sets described herein, the user can select a variety of labelsappropriate for the selected detection modality. As used herein, theterm “label” means a detectable moiety that generates a sufficientsignal to be registered by a device or instrument configured to read thedetectable moiety. The term “label” includes a detectable moietygenerated when an enzyme associated with a probe is contacted with asubstrate and converts the substrate into a detectable moiety thatgenerates a sufficient signal to be registered by a device or instrumentconfigured to read the detectable moiety. Exemplary labels includechromogenic, optical, fluorescent, chemiluminescent, magnetic,plasmonic, mass-based, electrochemical labels, and phenolic substrates(e.g., tyramine and tyrosine) acted upon by horseradish peroxidase.Accordingly, labels having a variety of chemical structures are usefulin the described methods and compositions. As used herein, the term“released label” means the portion of probe containing a label, whichhas been cleaved by the cleavage agent. As a consequence, thelabel-containing portion is partially or fully dissociated from itsparent molecule or complex.

In some embodiments, a released label contains a nucleotide sequencecapable of binding to a background-reducing agent containing acomplementary nucleotide sequence.

In an embodiment, a fluorescent label is used. General categories offluorescent labels include organic dyes, biological fluorophores,quantum dots, and nanoparticles including carbon dots. Specificfluorescent dyes include fluorescein, rhodamine, cyanine dyes, ALEXAdyes, DYLIGHT dyes, and ATTO dyes. The Examples herein describe use offour spectrally distinct fluorescent labels in a single round ofdetection. It is possible to use more than four spectrally overlappingfluorophores in one round of detection. Use of software to assist indetecting fluorophores having overlapping signals is known (see forexample, U.S. Pat. No. 6,750,964). A variety of fluorescent dyes andfilters are commercially available, allowing the methods describedherein to be performed using any feasible number of fluorescent labels.As described herein, in an embodiment, the methods can be performedusing a single fluorescent label; two fluorescent labels; threefluorescent labels; four fluorescent labels; five fluorescent labels;six fluorescent labels; seven fluorescent labels; eight fluorescentlabels; and greater than eight fluorescent labels. FIGS. 1A-1D depict aprobe-set that can be employed using a single fluorescent label becauseeach round of detection is independent. FIG. 2A depicts three probe-setswhere all probes of the first probe-set (A) have the same label; allprobes of the second probe-set (B) have the same label; and all probesof the third probe-set (C) have the same label. Thus, in an embodiment,the first and second (and optionally third and/or subsequent) detectablelabels are the same. In another embodiment, the first and second (andoptionally third and/or subsequent) detectable labels are different.

Generally, when using more than one fluorescent label, signals aredetected in different detection channels which correspond to differentregions of the light spectrum. Table 2 below shows four detectionchannels and representative popular fluorophores. If desired, aprobe-set can be designed for detection in a particular detectionchannel. Thus, a collection of probe-sets can be designed for detectionin different detection channels such that all first probes are detectedin a first detection channel; all second probes are detected in a seconddetection channel; and, when present, all third probes are detected in athird detection channel. Likewise for subsequent probes. In thisembodiment, the labels within a probe-set can be the same, or can bedifferent but detectable in the same detection channel. However, it isnot necessary for probe-sets to be aligned with each other in thismanner. Because the methods can employ a variety of labels, each roundof detection can employ a different detection channel or detectionmodality if desired. Using different modalities can expand the number oflabels available to the user when designing probes as described herein.

TABLE 2 Microscope Emission Detection Detection Wavelength Range Channel(nm) Example Fluorophores “FITC” 510-530 FITC, FAM, Fluorescein, Cy2,Alexa Fluor 488, Atto 488, “TRITC” 570-590 TRITC, TAMRA, Cy3, Quasar570, Alexa Fluor 568, Atto 550 “Cy5” 670-690 Cy5, Alexa Fluor 647, Atto647N, Quasar 670 “Cy7” 750-780 Cy7, Alexa Fluor 750, Atto 740, IRDye 750

Exemplary chromogenic labels include diaminobenzidine (DAB), nitro bluetetrazolium chloride (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP),and 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (X-Gal). A label canbe a moiety that operates through scattering, either elastic orinelastic scattering, such as nanoparticles and Surface Enhanced RamanSpectroscopy (SERS) reporters (e.g., 4-Mercaptobenzoic acid,2,7-mercapto-4-methylcoumarin). A label can also be achemiluminescence/electrochemiluminescence emitter such as rutheniumcomplexes and luciferases.

Results of Detection

Data outputs from detection of labels using a probe-set described hereincan be used to determine the presence, absence, and/or location oftarget molecules in a sample. The particular data output will depend onthe detection method. For example, when a fluorescence microscopy methodis used as described in the Examples below, software for aligningfluorescence microscopy images is well known and available fromcommercial and public sources (e.g. HALO software (Indica Labs), ZENsoftware (Zeiss), ImageJ: (imagej.nih.gov/ij/download.html).)

The methods described herein involve detecting targets in a “sample.” Asused herein, the term “sample” means any natural or man-made biologicalfluid, cell, tissue, or fraction thereof, or other material, thatincludes or is suspected to include a target. A sample can be derivedfrom a prokaryote or eukaryote and therefore can include cells from, forexample, animals, plants, or fungi. Accordingly, a sample includes aspecimen obtained from one or more individuals or can be derived fromsuch a specimen.

For example, a sample can be a tissue section obtained by biopsy, orcells that are placed in or adapted to tissue culture. Exemplary samplesinclude biological specimens such a cheek swab, amniotic fluid, skinbiopsy, organ biopsy, tumor biopsy, blood, urine, saliva, semen, sputum,cerebral spinal fluid, tears, mucus, and the like. A sample can befurther fractionated, if desired, to a fraction containing particularcell types. For example, a blood sample can be fractionated into serumor into fractions containing particular types of blood cells. Ifdesired, a sample can be a combination of samples from an individualsuch as a combination of a tissue and fluid. A sample can be, or cancontain, a laboratory preparation that includes or is suspected toinclude a target.

When used in a method described herein, an assay component such as asample, target-specific binding partner, background-reducing agent, orother element, can be attached to a surface. Exemplary surfaces includea slide, a plate, a bead, a tube, and a capillary. Examples 1 and 6-8describe examples of use of tissue samples attached to slides. Example 2describes an exemplary use of target-specific binding partners attachedto an assay plate. Example 3 describes an exemplary use oftarget-specific binding partners attached to a bead.

Prior to analysis, a sample can be processed to preserve the integrityof targets. Such methods include the use of appropriate buffers and/orinhibitors, including nuclease, protease and phosphatase inhibitors,that preserve or minimize changes in the molecules in the sample.Methods for preserving tissue samples are well known and includefixatives. The particular preservation method selected will depend onthe tissue or cell sample, and the molecular attributes of thetarget-specific binding partners selected.

As used herein, the term “cleavage site” with reference to a probedescribed herein means a structure within the probe that is susceptibleto the action of its corresponding cleavage agent. Upon contacting acleavage agent with its corresponding cleavage site, one or more bondsare cleaved. As used herein, to “cleave” a chemical bond encompasses oneor more of: breaking, isomerizing, and/modifying a covalent ornoncovalent bond. In some cases, cleavage of a cleavage site thereforeresults in production of a portion of the probe containing a label(“released label”). In other cases, cleavage may result in a bondmodification or isomerization that changes the label character so thatit is no longer substantially detected (it is “suppressed”). Thus, themolecular characteristics of a cleavage site depend on its correspondingcleavage agent. A cleavage site can be selected, for example, from achemical cleavage site; a mechanical cleavage site; an electromagneticcleavage site; an enzymatic cleavage site.

Therefore, a variety of cleavage agents are useful in the methods andcompositions described herein. A cleavage agent can be a chemical agent,enzymatic agent, electromagnetic agent (e.g., UV light, visible light,infrared, near infrared, x-ray, microwave, radio waves, gamma rays),mechanical force (including an acoustic force), or any other agent thatsuppresses or releases a probe segment, for example, by breaking,isomerizing, and/or chemically modifying a bond. The purpose of labelcleavage (leading to release or suppression) is to reduce the signal toa level that does not interfere with detection of subsequent labels, orthat is otherwise acceptable to the user. For example, when usingfluorescent labels, cleaving bonds of a fluorophore can destroy itsfluorescence or alter the wavelength at which its fluorescence issubstantially detected.

Exemplary chemical bonds that can be cleaved are disulfide bonds(cleaved by reducing agents such as dithiothreitol ortris(2-carboxyethyl)phosphine)), esters (cleaved by hydroxylamine),vicinal diols (cleaved by sodium meta-periodate), sulfones (cleavedunder basic conditions), photocleavable bonds (cleaved by light), andbonds that can be cleaved using enzymes such as proteases, hydrolases,nucleases, uracil DNA glycosylase, and DNA glycosylase-lyaseEndonuclease VIII, (e.g., USER (Uracil-Specific Excision Reagent) (NewEngland Biolabs). Non-natural nucleotides, amino acids, or othercompounds that serve as substrates for particular enzymes can be used ina cleavage site. For example, 8-oxoguanine may be cleaved by DNAglycosylase OGG1. For example, a 1′,2′-Dideoxyribose, dSpacer,apurinic/apyrimidinic, tetrahydrofuran, or abasic furan may be cleavedby Endonuclease VIII cleavage sites.

In an embodiment, an electromagnetic cleavage site is a photocleavagesite, which is cleaved by the presence of light of a particular spectralrange. A variety of photocleavable moieties can be used in the probesdescribed herein. A variety of chemical bonds are susceptible tophotocleavage (see, for example, Olejnik et al., Nucleic Acids Res. 1999Dec 1; 27(23):4626-31; and Leriche et al. Bioorganic & MedicinalChemistry, volume 20(2), 571-582 (2012).) Among well-knownphotocleavable moieties include o-nitrobenzyl (ONB) esters,α-thioacetophenone moieties, and 7-amino coumarin moieties. See, forexample, CRC Handbook of Organic Photochemistry and Photobiology, 2ndEdition, chapter 69. Exemplary photocleavable moieties that can beincorporated into oligonucleotides are commercially available throughBiosynthesis, Inc., Lewisville, Tex.; Integrated DNA Technologies,Coralville, Iowa, and other companies.

A variety of enzymatically cleavable moieties can be used in the probesdescribed herein. A number of enzymes can break the covalent bondswithin a nucleic acid molecule. For example, glycosylases can remove abase from the sugar moiety of a nucleotide; endonucleases, exonuclease,DNAzymes, and deoxyribozymes can cleave phosphodiester bonds of nucleicacid molecules, and enzymes can be engineered for cleaving at a cleavagesite within a probe described herein.

A glycosylase capable of specifically removing a base that participatesin nucleotide base-pairing can reduce the strength of interactionbetween the two strands. For example, deoxyuridine (dU) can besubstituted for deoxythymidine (dT) at a cleavage site; dU would pairwith dA, and this pair would be cleaved by Uracil-DNA Glycosylase (UDG,commercially available from New England Biolabs, Cat #M0280S). Thisreaction will result in abasic site(s) at the cleavage site. Such abasicsites can be further cleaved by Endonuclease VIII, or another method.This promotes dissociation of remnant binding pairs. UDG or acombination of UDG and Endonuclease VIII can therefore be useful inperforming the methods described herein. A mixture of these enzymes iscommercially available (e.g., from New England Biolabs, under thetradename USER, Cat#M5505S).

A UDG cleavage site useful in the described probes will contain a numberof dU nucleotides, ranging from 1 to 5 dUs; 1 to 10 dUs; 1 to 15 dUs;and 1 to 20 dUs. When using UDG and Endonuclease VIII, the dUs can beplaced in a way that, after removal of dU, the remnants are short (e.g.,less than or equal to about 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides)such that they dissociate spontaneously and relatively quickly. Whenusing UDG (i.e., no Endonuclease VIII) removal of dU units coulddestabilize the strand enough to separate a label from a quench moiety.

Endonucleases having site specific activity useful in the methodsdescribed herein include restriction endonucleases, zinc fingernucleases, transcription activator-like effector nucleases (TALENs), anddeoxyribozymes.

An RNA guided endonuclease can be used in methods described herein. Forexample, Cas9 (CRISPR associated protein 9) is an RNA-guidedendonuclease can specifically cleave an engineered cleavage site. Onestrand may be cleaved, for example using a nicking endonuclease. As anexample, Cas9 nickases are Cas9 enzymes that have been engineered toonly include one active cleaving site, leading to single strand cuts,while conserving the high specificity of Cas9.

A variety of protein cleavage chemistries can be used in methodsdescribed herein, including protein modification chemistries. Somemethods modify native amino acids while others require geneticmanipulation of the amino acid sequence before modification. Examplesinclude: Yu, Y. et al. Chemoselective peptide modification viaphotocatalytic tryptophan β-position conjugation. J. Am. Chem. Soc. 140,6797-6800 (2018); Willwacher, J., Raj, R., Mohammed, S. & Davis, B. G.Selective metal-site-guided arylation of proteins. J. Am. Chem. Soc.138, 8678-8681 (2016); Krall, N., da Cruz, F. P., Boutureira, O. &Bernardes, G. J. L. Site-selective protein-modification chemistry forbasic biology and drug development. Nat. Chem. 8, 103-113 (2016).

In an embodiment, a suitable cleavage agent is capable of breakingcovalent bonds within a probe to release a segment (where the segmentcan contain, for example, a label, quench moiety, or other unwantedsegment). Examples of cleavage agents and cleavage modes are shown inFIGS. 4A-4D as described above. One or more combination of differenttypes of cleavage agents and different types of cleavage sites may beused. For example, in a probe-set, one or more probes contain a cleavagesite for releasing a label (FIG. 4B); and one or more probes contain acleavage site for unquenching a label (FIG. 4D). One or more probes cancontain both a cleavage site for releasing a label and a cleavage sitefor unquenching a label. In an embodiment, a cleavage agent is capableof releasing a label (or labels) and unquenching a different label (orlabels). If desired, a combination of cleavage agents can be used in anystep in which a single cleavage agent is used.

Depending on the nature of the probes and cleavage agent used in aparticular step of a method described herein, the methods can involvewashing a sample after contacting the sample with a cleavage agent. Thisstep can be useful for removing unwanted signal from released labels;for removing cleavage agent used in a prior step. In an embodiment,washing is not needed. For example, washing is not needed when thesignal of the released label does not interfere with the user'sexperimental intent, such as when the label is suppressed upon treatmentby the cleavage agent; or the released label diffuses way or otherwisedoes not create significant background. Therefore, in certainembodiments, when performing the method on a sample underneath acoverslip or other enclosure, it is not necessary to remove or otherwisedisturb the enclosure when carrying out one or more steps of the method.As described below, a background-reducing agent can be employed whencarrying out methods without directly accessing a sample, for example,without removing a coverslip.

As used herein, the term “quench moiety” means any physical or chemicalcharacteristic of a probe that functions to quench one or more labels ofthe probe. The signal produced by a label is quenched when the signal isreduced as compared with the signal in the absence of the quench moietyby at least 10%, for example at least 15%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, at least 99%, and completelyquenched. The level of signal that renders a label undetectable ismethod-dependent. Thus, the term “undetectable” means a level of signalthat is acceptable to the user for the method employed. For example, anundetectable signal produces a signal-to-background ratio that permitsdetection of a desired signal without interference.

When the label is a fluorophore, the quench moiety reduces the emissionfrom the fluorophore when attached to or in proximity to thefluorophore. Any of a variety of fluorophore quench moieties can be usedin a method described herein. Exemplary quench moieties include, but arenot limited to, Dabacyl, Cy50, Iowa Black, OXL570, BHQ-I, BHQ-2, BHQ-3,and Si-rhodamine based quenchers. Label-Quencher pairs are well known.Exemplary fluorophores and spectrally compatible quenchers includeCoumarin and Dabacyl; ALEXA Fluor 488 and BHQ-1; Cy3 and Iowa Black RQ;TAMRA and OXL570; and IRDye 680 and BHQ-3.

The location of a quench moiety relative to a label will depend on theparticular physical and chemical characteristics of the componentsselected. For a nucleic acid probe, a quench moiety can be attached, forexample, on the end of a nucleotide of a probe, internally, on abranched nucleotide segment, or other location so long as the quenchmoiety is in sufficient proximity to the label. For example, a quenchmoiety can be attached at the phosphate moiety of the 3′ nucleotide of aprobe.

A probe can contain a tertiary structure that maintains a label andquencher in close proximity to achieve quenching. Upon treatment with acleavage agent, such a probe would undergo a conformation change thatseparates the label and quench moiety to de-quench the label. Forexample, a nucleic acid hairpin can be destabilized by treatment withuracil-DNA glycosylase, increasing the distance between fluorophore andquench moiety, thereby de-quenching the signal from the fluorophore. Fora polypeptide probe, a variety of tertiary structures can be engineeredinto a polypeptide to achieve a desired distance between a label andquench moiety (see, for example, Chen et al., Can. J. Chem. 93: 389-398(2015)).

FIG. 2A illustrates three exemplary probe-sets in which all secondprobes (B) contain the same quench moiety; and all third probes (C)contain the same quench moiety. This represents the concept that labelsare paired with quench moieties that function effectively to render thelabel undetectable.

The probes and target-specific binding partners described herein can beprepared using standard molecular biology and chemical methods fordesigning molecules containing two functional sites or joining two ormore molecules together to acquire the required functionality. A methodfor preparing target-specific binding partners that are antibodies withcovalently attached nucleic acid strands is described in Wang et al.Nano Lett., 17, 6131-6139 (2017). The procedure involves crosslinking ofthiol-modified DNA oligonucleotides to lysine residues on antibodies, asdescribed in Agasti et al, Chem Sci, 8, 4, 3080-3091 (2017). In brief,250 uM 5′ thiol-modified DNA oligonucleotides (Integrated DNATechnologies) were activated by 100 mM DTT for 2 hours and then purifiedusing NAP5 columns (GE Healthcare Life Sciences, 17-0853-02) to removeexcessive DTT. Antibodies formulated in PBS were concentrated using 100KDa Amicon Ultra Filters (EMDMillipore, UFC510096) to 2 mg/ml andreacted with maleimide-PEG2-succinimidyl ester crosslinkers (Sigma746223) for 2 hours. Antibodies were then purified using 0.5 ml 7 kDAZeba desalting columns (LifeTechnologies, 89883) to remove excessivecrosslinkers. Activated DNA oligonucleotides were incubated withantibodies (11:1 DNA: Antibody ratio) overnight at 4° C. Finalconjugated antibodies were washed using PBS/BSA (100 ug/ml) using AmiconUltra Filters four times to remove nonreacted DNA oligonucleotides. Analternative method is to employ the SiteClick kit from ThermoFisher(S10467).

The skilled artisan will understand that the figures, described above,and example, described below, are for illustration purposes only.Neither the figures nor the examples are intended to limit the scope ofthe disclosed teachings in any way.

EXAMPLES

The following examples are provided to illustrate certain disclosedembodiments and are not to be construed as limiting the scope of thisdisclosure in any way.

Example 1

This example shows detection of eight distinct targets on a singleformalin-fixed paraffin-embedded (FFPE) tonsil slide using aTris(2-carboxyethyl)phosphine (TCEP) based cleavage mode.

A human tonsil tissue slide (Amsbio LLC, Cambridge, Mass.) was firstbaked for 30 min at 60° C. and then deparaffinized in a GEMINI automatedslide stainer (ThermoFisher). The slide was then washed three times inPBS, before blocking with the Ultivue antibody diluent solution(UltiMapper™ I/O kits, Ultivue, Cambridge, Mass.) for 15 min at roomtemperature. Eight different antibodies (selective for CD45RO, PD1, CD3,Cytokeratin, CD8, CD68, PD-L1 and Ki67) conjugated with distinct DNAbarcodes were together added to the tissue slide and incubated for 1 hr.at room temperature (see Table 1 for exemplary nucleic acid bar codes).The slide was then washed three times in PBS, followed by incubationwith Ultivue pre-amplification mix (UltiMapper™ I/O kits) for 25 min. atroom temperature. The slide was then washed three times in PBS; Ultivueamplification solution (UltiMapper™ I/O kits) was the added to the slideand incubated for 90 min. at 30° C. in a hybridization oven (SLIDE MOAT,Boekel, Feasterville, Pa.). The slide was then washed three times in PBSfollowed by incubation with Ultivue nuclear counterstain (UltiMapper™I/O kits) for 15 min. at room temperature in a dark environment. Theslide was then washed three times in PBS. A cocktail containing alleight fluorescent probes (probe-set design as shown in FIGS. 1A-1B)diluted in Ultivue probe buffer (UltiMapper™ I/O kits) was added to theslide and incubated for 25 min. at room temperature in a darkenvironment. The slide was then washed three times in PBS, mounted inPBS and coverslipped.

The whole tissue fluorescence image for round 1 was then acquired on aPerkinElmer Polaris microscope at 20× magnification. Following theacquisition of the round 1 image, the slide was de-coverslipped andincubated with about 10 molar equivalents TCEP for 15 minutes at roomtemperature. The slide was washed three times in PBS, mounted in PBS andcoverslipped. The whole tissue fluorescence image for round 2 was thenacquired at 20× magnification.

FIGS. 6A-6B show results demonstrating detection of eight unique proteintargets on a single FFPE tonsil slide using TCEP based cleavage mode intwo detection rounds (FIGS. 6A and 6B; four targets for each round)without the addition of probes between the detection rounds. Probes forCD45RO, PD1, CD3 and Cytokeratin, have a disulfide cleavage site betweenthe barcode recognizing domain and the respective fluorescent dyes.Probes for CD8, CD68, PDL1 and Ki67 have a disulfide cleavage sitebetween the respective fluorescent dyes and the quench moieties (see,for example, FIG. 4B for a schematic).

The fluorescent dyes were selected to be detected in four differentdetection channels: FITC, TRITC, Cy5 and Cy7 (see Table 2). Specificsignal was observed for CD45RO, PD1, CD3 and Cytokeratin targets inround 1 of detection (FIG. 6A). No detectable signal is observed forCD8, CD68, PDL1 and Ki67 targets in round 1 of detection (FIG. 6A) asthe fluorescence signal is initially suppressed by the quench moiety.Post-TCEP treatment, which facilitates simultaneous release of thefluorescent dye and de-quenching of the specific signal, the specificsignal is observed for CD8, CD68, PDL1 and Ki67 targets in round 2 ofdetection (FIG. 6B). No detectable signal is observed for CD45RO, PD1,CD3 and Cytokeratin targets in round 2 of detection.

Thus, a probe-set as described herein was useful for detecting eighttargets in two rounds of detection.

Example 2

This example describes detection of eight unique targets in aplate-based assay using a TCEP based cleavage mode.

Nunc MaxiSorp (Thermo Fisher) microplate wells are coated with a mixtureof capture antibodies (Abcam) to Human IFN alpha, IL-6, TNF alpha, IFNgamma, IFN beta, IFN lambda, IL-1a, and IL-4 at a total antibodyconcentration of 5 μg/ml in 10mM phosphate buffered saline, pH 7.2 (PBS)for 2 hr at ambient temperature. The wells are aspirated and blockedwith PBS containing 2% bovine serum albumin overnight at ambienttemperature. The wells are aspirated and samples and standards are addedfor 2 hr at 37° C. The wells are washed with PBS containing 0.05%Tween-20 (PBST). A mixture of eight target-specific binding partners,which are antibodies to Human IFN alpha, IL-6, TNF alpha, IFN gamma, IFNbeta, IFN lambda, IL-1a, and IL-4 conjugated with unique DNA barcodes(see Table 1 for exemplary nucleic acid bar codes), is added to eachwell for 1 hr. at ambient temperature. The wells are washed with PBST,followed by incubation with Ultivue pre-amplification mix (UltiMapper™I/O kits) for 25 min. at ambient temperature. The wells are washed withPBST and the DNA barcodes are amplified as described in WO2018/107054,which is incorporated herein by reference. The wells are washed withPBST and a cocktail containing all eight fluorescent probescomplimentary to the DNA barcodes (Human IFN alpha, IL-6, TNF alpha, IFNgamma as the first probe (A) in FIG. 1A; IFN beta, IFN lambda, IL-1a,and IL-4 as a probe similar to the second probe (B) in FIG. 1B butwithout the second cleavage site (C₂)) diluted in Ultivue probe buffer(UltiMapper™ I/O kits) is added and incubated for 25 min. at ambienttemperature in a dark environment. The wells are washed with PBST, andfirst round of fluorescence detection (Human IFN alpha, IL-6, TNF alpha,IFN gamma) is performed on a Synergy H1 Microplate Reader (BioTek).

Following fluorescence detection of the first round signals, TCEP isadded and incubated for 15 min. at ambient temperature, which removesthe first round signals and exposes the second round signals. The wellsare washed with PBST and the second round of fluorescence detection (IFNbeta, IFN lambda, IL-1a, and IL-4) is performed.

Controls containing standard concentrations of analytes are employed, tothat concentrations of target molecule can be determined if desired. Theresults provide an 8-plex analysis of the presence or absence, orconcentrations of, the targets in each sample.

Example 3

This example describes detection of eight unique targets in a bead-basedimmunoassay using a TCEP based cleavage mode.

Capture antibodies for each target are immobilized on Dynabeads, M-270epoxy, 2.8 μm in diameter (ThermoFisher) according to the manufacturer'sinstructions. The beads are blocked and incubated with target-specificbinding partners and a probe-set essentially as described in Example 2.Fluorescence detection of first round signals is performed on a flowcytometer, followed by TCEP incubation and detection of second roundsignals.

Example 4

This example describes detection of a target using a probe with a uracilDNA glycosylase cleavage agent.

This example shows use of a probe as depicted in FIGS. 7A-7B, rightpanel. A sample was prepared essentially as described in Example 1, butfor a single target, CD3. The sample was incubated with target-specificbinding partner for CD3 and a probe containing a unique nucleic acidbarcode; a fluorescent label, and a corresponding quench moiety (FIGS.7A-7B, right panel).

The sample was imaged to detect the fluorescent label. FIG. 7A showsthat no signal was detected, demonstrating that the label was quenched.The sample was then treated with uracil DNA glycosylase (New EnglandBiolabs; 0.1 Units/ul Uracil DNA Glycosylase in 1× Cut Smart Bufferincubated on the tissue sample at 37C for 15 mins. (seewww.neb.com/products/b7204-cutsmart-buffer#Product%20Information) andre-imaged. FIG. 7B shows that the label was unquenched, therebyactivating signal from the label.

Example 5

This example describes detection of three miRNA targets using a TCEPbased cleavage mode.

Target-specific binding partners for miRNA targets contain DNA sequencesthat recognize the particular miRNAs. For example, a DNA sequenceselective for miR-146a is 5′-AACCCATGGAATTCAGTTCTCA-3′ (SEQ ID NO: 25);a DNA selective for miR-15a is 5′-CACAAACCATTATGTGCTGCTA-3′ (SEQ ID NO:26); and a DNA sequence selective for MiR-155 is 5′CCCCTATCACGATTAGCATTAA-3′ (SEQ ID NO: 27). These sequences areincorporated into a target-specific binding partner that contains aunique barcode (see, for example, Table 1) for each target.Corresponding probes can be designed to contain sequences complementaryto the barcodes (Table 1), and label, quencher, and cleavage sitecompositions that function to permit sequential detection (see e.g.,FIGS. 1A-1D).

The paraffin section is baked at 65° C. for 1 hr, and deparaffinize inxylene (2×10 minutes), rehydrated in ethanol solutions (100%, 90%, 80%,70%), and DEPC-treated water and PBS wash. The section is incubated withProteinase K (20 ug/mL) at 37° C. for 10 min., washed with PBS and fixedwith 4% paraformaldehyde for 10 min. It is then washed with PBS, 100 mMglycine, PBS, and 2×SSC (diluted from 20×, ThermoFisher). The section isthen pre-hybridized in a solution of 50% deionized formamide, 2×SSC,1×Denhardt's, 0.02% SDS, yeast tRNA (0.5mg/mL), and salmon sperm DNA(0.5mg/mL) for 2 hr at 50° C.

The probes are prepared with DNA barcodes on the 3′ ends. These arehybridized overnight at 50° C. in a solution of 50% deionized formamide,2×SSC, 1×Denhardt's, 10% dextran sulfate, yeast tRNA (0.5 mg/mL), andsalmon sperm DNA. The section is washed with 2×SSC at 37° C., 2×SCC at50° C., 1×SSC at 37° C. 1×SCC at 50° C., 0.02% SDS in 1×SSC at 37° C.,1×SSC at 50° C., and PBST at ambient temperature. The section is washedwith PBST, followed by incubation with Ultivue pre-amplification mix for25 min. at ambient temperature. The section is washed with PBST andUltivue amplification solution is added and incubated for 90 min. at 30°C.

A cocktail containing all fluorescent probes (some prepared as in thefirst probe (A) in FIG. 1A and others prepared as the second probe (B)in FIG. 1B but without the second cleavage site (C₂)), complimentary tothe DNA barcodes, is added and incubated for 25 min. at ambienttemperature in a dark environment. The first round of fluorescencedetection; TCEP incubation; and second round of fluorescence detectionare performed essentially as described in Example 1.

Example 6

This example describes detection of eight unique targets on a singleformalin-fixed paraffin-embedded (FFPE) tonsil slide using aphotocleavage method without coverslip removal.

A human tonsil tissue slide (Amsbio LLC, Cambridge, Mass.) was firstbaked for 30 min at 60° C. and then processed on a Leica BONDRxautostainer. The slide was deparaffinized using the dewax solution(AR9222, Leica Biosystems) with 4-step dewax protocol and antigenretrieved by incubating epitope retrieval solution 2 (AR9640, LeicaBiosystems) for 20 minutes at 100° C. The slide was then washed threetimes with the Leica wash solution (AR9590, Leica Biosystems), beforeblocking with the Ultivue antibody diluent solution (UltiMapper™ I/Okits, Ultivue, Cambridge, Mass.) for 15 min at room temperature.

Eight different antibodies (CD8, PD1, PDL1, CD68, CD3, CD4, FoxP3 andCytokeratin) conjugated with unique DNA barcodes were together added tothe tissue slide and incubated for 1 hr at room temperature. The slidewas then washed three times with Leica wash solution, followed byincubation with Ultivue pre-amplification mix (UltiMapper™ I/O kits) for25 min at room temperature. The slide was then washed two times withLeica wash solution followed by incubating the slide with Leica washsolution for 5 min at 35° C. Ultivue amplification solution (UltiMapper™I/O kits) was then added to the slide and incubated for 90 min at roomtemperature. The slide was then washed three times with Leica washsolution followed by incubation with Ultivue nuclear counterstain(UltiMapper™ I/O kits) for 15 min at room temperature. The slide wasthen washed three times with Leica wash solution.

Two probe-sets of four probes each were used. A cocktail containing alleight fluorescent probes (0.5 to 2 μM) diluted in Ultivue probe buffer(UltiMapper™ I/O kits) was added to the slide and incubated for 25 minat room temperature. Probes for CD8, PD1, PDL1 and CD68 (Probe design Ain FIG. 11A), contain a UV cleavage site between their barcodecomplementary regions and the respective fluorescent dyes. The portionof the probe released upon cleavage contains a region complementary to abackground-reducing agent present in the mounting medium applied to thesample prior to imaging. Probes for CD3, CD4, FoxP3 and Cytokeratin,(Probe design B in FIG. 11A), contain a UV-activated cleavage sitebetween the respective fluorescent dye and the quencher. The slide wasthen washed three times in Leica wash solution, mounted in mountingmedia containing activatable background-reducing agents (100-800 nM)corresponding to each first probe (see FIG. 11A for background-reducingagent design, indicating that the background-reducing agent containedtwo cleavage sites) and coverslipped with UV clear coverslips.

The fluorescence image for round 1 was then acquired on a Zeiss AxioScanZ1 microscope at 20× magnification. Following the acquisition of theround 1 image, the entire tissue was then scanned using the Colibri7 385nm LED with a 10× objective with 5 secs exposure to photocleave thecleavage sites on the first probes and corresponding background-reducingagents. The fluorescence image for round 2 was then acquired at 20×magnification immediately.

FIGS. 11B-11C show fluorescence signals from the eight unique proteintargets. Specific signal observed for CD8, PD1, PDL1 and CD68 targets inround 1 of detection (FIG. 11B). No detectable signal observed for CD3,CD4, FoxP3 and Cytokeratin targets in round 1 of detection (FIG. 11C) asthe fluorescence signal is initially suppressed by the quench moiety.Use of UV light as a cleavage agent facilitates simultaneous release ofthe fluorescent dye for Probe design A (FIG. 11A) and de-quenching ofProbe design B (FIG. 11A). Specific signal was observed for CD3, CD4,FoxP3 and Cytokeratin targets in round 2 of detection (FIG. 11C). Nodetectable specific signal observed for CD8, PD1, PDL1 and CD68 targetsin round 2 of detection. Additionally, no detectable fluorescent signalobserved in round 2 of detection from the released fluorescent dyes.

Example 7

This example shows the use of background-reducing agents to reducefluorescence signal from probe labels released from round A probes afterphotocleavage and without coverslip removal.

Two human tonsil tissue slides (Amsbio LLC, Cambridge, Mass.) were firstbaked for 30 min at 60° C. and then processed on a Leica BondRxautostainer. The slides were deparaffinized using the dewax solution(AR9222, Leica Biosystems) with 4-step dewax protocol and antigenretrieved by incubating epitope retrieval solution 2 (AR9640, LeicaBiosystems) for 20 minutes at 100° C. The slides were then washed threetimes with the Leica wash solution (AR9590, Leica Biosystems), beforeblocking with the Ultivue antibody diluent solution (UltiMapper™ I/Okits, Ultivue, Cambridge, Mass.) for 15 min at room temperature. CD3antibody conjugated with DNA barcodes was added to the tissue slides andincubated for 1 hr at room temperature. The slides were then washedthree times with Leica wash solution, followed by incubation withUltivue pre-amplification mix (UltiMapper™ I/O kits) for 25 min at roomtemperature. The slides were then washed two times with Leica washsolution followed by incubating the slide with Leica wash solution for 5min at 35° C. Ultivue amplification solution (UltiMapper™ I/O kits) wasthen added to the slides and incubated for 90 min at room temperature.The slides were then washed three times with Leica wash solutionfollowed by incubation with Ultivue nuclear counterstain (UltiMapper™I/O kits) for 15 min at room temperature. The slides were then washedthree times with Leica wash solution. Fluorescent probe (probe design asshown in FIGS. 12A, 12C) diluted in Ultivue probe buffer (UltiMapper™I/O kits) was added to the slides and incubated for 25 min at roomtemperature. The slides were then washed three times in Leica washsolution. One of the tissue slides was then mounted in the 1× TAE Mg2+buffer, whereas the other slide was mounted in mounting media containingbackground-reducing agent in 1× TAE Mg2+ buffer. Both tissues slideswere coverslipped with UV clear coverslips.

The whole tissue fluorescence image for round 1 was then acquired on aZeiss AxioScan Z1 microscope at 20× magnification. Following theacquisition of the round 1 image, the entire tissue was then scannedusing the Colibri7 385 nm LED with a 10× objective with 5 secs exposureto photocleave the cleavage sites on the first probes. The whole tissuefluorescence images were then acquired at 20× magnification immediatelyafter photocleavage.

Specific signal was observed for CD3 for both slides (FIGS. 12A and 12C)before photocleavage. No detectable initial quenching was observed forthe slide mounted in the presence of photoactivatablebackground-reducing agent when comparing the signal between the twoslides before cleavage. Residual diffused fluorescent signal wasobserved for the slide (FIG. 12B) in the absence of background-reducingagent in mounting media (see FIG. 12C for background-reducing agentdesign) after cleavage without coverslip removal. Minimal to nodetectable residual fluorescent signal was observed for the slide withbackground-reducing agent in mounting media (see FIG. 12D) aftercleavage without coverslip removal.

Example 8

This example describes detection of sixteen unique targets on a singleformalin-fixed paraffin-embedded (FFPE) tonsil slide using threeprobe-sets and three different cleavage agents.

A human tonsil tissue slide (Amsbio LLC, Cambridge, Mass.) was firstbaked for 30 min at 60° C. and then processed on a Leica BONDRxautostainer. The slide was deparaffinized using the dewax solution(AR9222, Leica Biosystems) with 4-step dewax protocol and antigenretrieved by incubating epitope retrieval solution 2 (AR9640, LeicaBiosystems) for 20 minutes at 100° C. The slide was then washed threetimes with the Leica wash solution (AR9590, Leica Biosystems), beforeblocking with the Ultivue antibody diluent solution (UltiMapper™ I/Okits, Ultivue, Cambridge, Mass.) for 15 min at room temperature.

Sixteen different antibodies (CD8, CD68, PDL1, Ki67, CD45RO, PD1, CD3,Cytokeratin, CD11c, GranzymeB, FoxP3, Lag3, CD20, CD163, CD4 and MHCII)conjugated with unique DNA barcodes were together added to the tissueslide and incubated for 1 hr at room temperature. The slide was thenwashed three times with Leica wash solution, followed by incubation withUltivue pre-amplification mix (UltiMapper™ I/O kits) for 25 min at roomtemperature. The slide was then washed two times with Leica washsolution followed by incubating the slide with Leica wash solution for 5min at 35° C. Ultivue amplification solution (UltiMapper™ I/O kits) wasthen added to the slide and incubated for 90 min at room temperature.The slide was then washed three times with Leica wash solution followedby incubation with Ultivue nuclear counterstain (UltiMapper™ I/O kits)for 15 min at room temperature. The slide was then washed three timeswith Leica wash solution. A cocktail containing all 16 fluorescentprobes diluted in Ultivue probe buffer (UltiMapper™ I/O kits) was addedto the slide and incubated for 25 min at room temperature.

Probes for CD8, CD68, PDL1 and Ki67 have a di-sulfide cleavage sitebetween the probe binding domain and the respective fluorescent dyes(FIG. 13A, probe design 1). Probes for CD45RO, PD1, CD3 and Cytokeratinhave a UV-cleavage site between the probe binding domain and therespective fluorescent dyes, and a di-sulfide cleavage site betweenfluorescent dye and corresponding quenchers (FIG. 13A, probe design 2).

Probes for CD11c, Granzyme B, FoxP3 and LAG3 have uracil bases in theprobe binding domain, and a UV-cleavage site between fluorescent dye andcorresponding quenchers (FIG. 13A, probe design 3). Probes for CD20,CD163, CD4 and MHCII have a hairpin structure consisting ofuridine-adenine base pairs between fluorescent dye and correspondingquenchers (FIG. 13A, probe design 4).

The slide was then washed three times in PBS, mounted in Prolong GoldAntifade Mountant (P36930, Thermo Fisher) and coverslipped. Thefluorescence image for round 1 was then acquired on a Zeiss AxioScan Z1microscope at 20× magnification. Following the acquisition of the round1 image, the slide was then de-coverslipped and incubated with TCEP for15 minutes. The slide was then washed three times in PBS, mounted inProlong Gold Antifade Mountant and coverslipped with UV clearcoverslips. The fluorescence image for round 2 was then acquired on aZeiss AxioScan Z1 microscope at 20× magnification. Following theacquisition of the round 2 image, the entire tissue was then scannedusing the Colibri7 385 nm LED with a 10× objective with 5 secs exposureto affect photocleavage. The slide was then de-coverslipped and washedthree times in PBS, mounted in Prolong Gold Antifade Mountant andcoverslipped. The whole tissue fluorescence image for round 3 was thenacquired on a Zeiss AxioScan Z1 microscope at 20× magnification.Following the acquisition of the round 3 image, the slide was thende-coverslipped and incubated with UDG enzyme for 10 minutes at 37° C.The slide was then washed three times in PBS, mounted in Prolong GoldAntifade Mountant and coverslipped. The fluorescence image for round 4was then acquired on a Zeiss AxioScan Z1 microscope at 20×magnification.

FIG. 13B shows detection of sixteen distinct protein targets on a singleFFPE tonsil slide using three different cleavage methods (TCEP, UV andUDG enzyme) in four detection rounds without the addition of probesbetween the detection rounds. Specific signal observed for CD8, CD68,PDL1 and Ki67 (FIG. 13A, probe design 1) targets in round 1 of detection(FIG. 13B, Round 1) and no detectable signal observed from other targetswith other probe designs. Specific signal observed for CD45RO, PD1, CD3and Cytokeratin (FIG. 13A, probe design 2) targets in round 2 ofdetection (FIG. 13B, Round 2) after TCEP chemistry and no detectablesignal observed from other targets with other probe designs. Specificsignal observed for CD11c, Granzyme B, FoxP3 and LAG3 (FIG. 13A, probedesign 3) targets in round 3 of detection (FIG. 13B, Round 3) after UVphotocleavage and no detectable signal observed from other targets withother probe designs. Specific signal observed for CD20, CD163, CD4 andMHCII (FIG. 13A, probe design 4) targets in round 4 of detection (FIG.13B, Round 4) after UDG enzyme treatment and no detectable signalobserved from other targets with other probe designs.

Example 9 Additional Embodiments

The following numbered items provide additional support for anddescriptions of the embodiments herein.

-   Item 1. A method for detecting a plurality of target molecules, the    method comprising:-   (a) contacting a sample with two or more target-specific binding    partners, wherein each target-specific binding partner comprises a    nucleic acid barcode; and is specific for a different target    molecule;-   (b) contacting the sample with one or more probe-sets wherein each    probe in a probe-set is specific for a different target-specific    binding partner, and wherein each probe-set comprises:-   a first probe, comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a first label; and    -   a cleavage site for a first cleavage agent, wherein the first        cleavage agent is capable of releasing the first label, and-   a second probe comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a second label;        -   a quench moiety, wherein the quench moiety renders the            second label undetectable; and    -   a cleavage site for the first cleavage agent, wherein the first        cleavage agent is capable of releasing the quench moiety,        whereby the second label is rendered detectable; and optionally        comprises a cleavage site for a second cleavage agent wherein        the second cleavage agent is capable of releasing the second        label;-   (c) detecting signals corresponding to labels of the first probes of    each of the one or more probe-sets;-   (d) contacting the sample with a first cleavage agent, thereby-   releasing the labels of the first probes in each of the one or more    probe-sets; and-   releasing the quench moieties of the second probes in each of the    one or more probe-sets, thereby activating signals corresponding to    the second labels, and-   (e) detecting signals corresponding to the labels of the second    probes of each of the one or more probe-sets.-   Item 2. The method of Item 1, wherein one or more of the probe-sets    further comprises a third probe, and the method further comprises:-   (f) in step (b), contacting the sample with the third probe    comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a third label; and    -   a quench moiety, wherein the quench moiety renders the third        label undetectable; and    -   a cleavage site for the second cleavage agent, wherein the        second cleavage agent is capable of releasing the quench moiety,        whereby the third label is rendered detectable;    -   and optionally comprises a distinct cleavage site for a distinct        cleavage agent capable of releasing the third label; and-   (g) after step (e), contacting the sample with a second cleavage    agent, thereby releasing the labels of the second probes in each of    the one or more probe-sets; and releasing the quench moieties of the    third probes in one or more probe-sets, thereby activating signals    corresponding to the third labels; and-   (h) detecting signals corresponding to the labels of the third    probes of one or more probe sets.-   Item 3. The method of Item 2, wherein one or more of the probe-sets    further comprise a subsequent probe, and the method further    comprises:-   (i) in step (b), contacting the sample with a subsequent probe    contained in one or more probe-set, wherein the subsequent probe    comprises:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a subsequent label; and    -   a quench moiety, wherein the quench moiety renders the        subsequent label undetectable; and    -   a cleavage site for a subsequent cleavage agent, wherein the        subsequent cleavage agent is capable of releasing the quench        moiety, whereby the subsequent label is rendered detectable;    -   and optionally comprises a distinct cleavage site for a distinct        cleavage agent capable of releasing activated labels from probes        in the sample;-   (j) contacting the sample with a subsequent cleavage agent, thereby    releasing activated labels of the probes in each probe-set; and    releasing the quench moieties of the subsequent probes in each    probe-set, thereby activating signals corresponding to the labels of    the subsequent probes in each probe-set; and-   (k) detecting signals corresponding to the labels of the subsequent    probes of each probe-set; and-   (l) optionally repeating steps (i) through (k).-   Item 4. The method of any one of Items 1-3, wherein the first and    second detectable labels of a probe-set are the same.-   Item 5. The method of any one of Items 1-3, wherein the first and    second detectable labels of a probe-set are different.-   Item 6. The method of any one of Items 2-5, wherein the two or more    of the first, second, and third detectable labels of a probe-set are    the same.-   Item 7. The method of any one of Items 2-5, wherein the two or more    of the first, second, and third detectable labels of a probe-set are    different.-   Item 8. The method of any one of Items 3-7, wherein two or more of    the first, second, third, and subsequent labels are the same.-   Item 9. The method of any one of Items 3-7, wherein two or more of    the first, second, third, and subsequent labels are different.-   Item 10. The method of any one of Items 1-9, further comprising    washing the sample after contacting the sample with the first    cleavage agent and/or after contacting the sample with the second    cleavage agent.-   Item 11. The method of any one of Items 1-9, wherein the sample is    not washed after contacting the sample with the first cleavage agent    and/or after contacting the sample with the second cleavage agent.-   Item 12. The method of Item 11, wherein the coverslip is not    removed.-   Item 13. The method of any one of Items 1-12, further comprising    increasing the number of nucleic acid barcodes on a target-specific    binding partner, wherein multiple copies of a corresponding probe    bind to multiple copies of the nucleic acid barcode.-   Item 14. The method of Item 13, wherein the number of nucleic acid    bar codes is increased using rolling circle amplification, primer    exchange reaction, hybridization chain reaction, or DNA branching.-   Item 15. The method of Item 14, wherein the number of nucleic acid    bar codes is increased before the target-specific binding partner is    contacted with the sample.-   Item 16. The method of Item 14, wherein the number of nucleic acid    bar codes is increased when the target-specific binding partner is    bound to its target molecule.-   Item 17. The method of any one of Items 1-16, wherein the released    label of a first probe comprises a nucleotide sequence.-   Item 18. The method of Item 17, further comprising contacting the    sample with a background-reducing agent comprising a nucleotide    sequence complementary to that of the released label of the released    first probe, wherein binding of the background-reducing agent to the    released label of the released first probe quenches the signal of    the label.-   Item 19. The method of any one of Items 2-18, wherein the released    label of a second probe comprises a nucleotide sequence.-   Item 20. The method of Item 19, further comprising contacting the    sample with a background-reducing agent comprising a nucleotide    sequence complementary to that of the released label of the second    probe, wherein binding of the background-reducing agent to the label    of the released second probe quenches the signal of the label.-   Item 21. The method of any one of Items 3-20, wherein an activated    label of a released probe comprises a nucleotide sequence.-   Item 22. The method of any one of Items 1-21, further comprising    contacting the sample with a background-reducing agent comprising a    nucleotide sequence complementary to that of the released label of    the probe, wherein binding of the background-reducing agent to the    released label of the released probe quenches the signal of the    label.-   Item 23. A probe-set composition comprising:-   one or more first probes, each comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a first label; and    -   a cleavage site for a first cleavage agent, wherein the first        cleavage agent is capable of releasing the first label, and-   one or more second probes, each comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a second label;    -   a quench moiety, wherein the quench moiety renders the second        label undetectable; and    -   a cleavage site for the first cleavage agent, wherein the first        cleavage agent is capable of releasing the quench moiety,        whereby the second label is rendered detectable;    -   and optionally comprises a distinct cleavage site for a distinct        cleavage agent capable of releasing the second label.-   Item 24. The composition of Item 23, further comprising:-   one or more third probes, each comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a third label;    -   a quench moiety, wherein the quench moiety renders the third        label undetectable; and    -   a cleavage site for a second cleavage agent, wherein the second        cleavage agent is capable of releasing the quench moiety,        whereby the third label is rendered detectable;    -   and optionally comprises a distinct cleavage site for a distinct        cleavage agent capable of releasing the third label,-   wherein the second probe further comprises a cleavage site for the    second cleavage agent.-   Item 25. The composition of Item 24, further comprising:-   a subsequent probe comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a subsequent label;    -   a quench moiety for the subsequent label, wherein the quench        moiety renders the subsequent label undetectable; and    -   a cleavage site, wherein a cleavage agent is capable of        releasing the quench moiety, whereby the subsequent label is        rendered detectable;    -   and optionally comprises a distinct cleavage site wherein a        distinct cleavage agent is capable of releasing the subsequent        label,-   wherein another probe in the probe-set comprises a cleavage site for    the same cleavage agent that releases the quench moiety of the    subsequent probe.-   Item 26. The composition of any one of Items 23-25, wherein the one    or more first probes have the same label.-   Item 27. The composition of any one of Items 23-25, wherein the one    or more first probes have a different label.-   Item 28. The composition of any one of Items 23-27, wherein the    cleavage site is an electromagnetic cleavage site; a chemical    cleavage site; or a mechanical cleavage site.-   Item 29. The composition of Item 28, wherein the electromagnetic    cleavage site is a photocleavage site.-   Item 30. The composition of Item 29, wherein the photocleavage site    is an ultraviolet (UV) cleavage site.-   Item 31. The composition of any one of Items 23-30, wherein the    first or second label is a fluorescent label.-   Item 32. The composition of any one of Items 23-31, further    comprising: a background reducing agent comprising a nucleotide    sequence complementary to a nucleotide sequence of a portion of a    first probe present between the cleavage site and the first label.-   Item 33. The composition of any one of Items 23-32, further    comprising: a background reducing agent comprising a nucleotide    sequence complementary to a nucleotide sequence of a portion of a    second probe present between the cleavage site and the second label.-   Item 34. The composition of any one of Items 25-33, further    comprising: a background reducing agent comprising a nucleotide    sequence complementary to a nucleotide sequence of a portion of a    subsequent probe present between the cleavage site and the    subsequent label.-   Item 35. A kit, comprising:-   the probe-set composition of any one of Items 23-34;-   a background-reducing agent;-   a coverslip;-   one or more target-specific binding partners;-   one or more buffers;-   one or more reagents for increasing the number of nucleic acid    barcodes of a target-specific binding partner;-   one or more cleavage agents;-   a nuclear counterstain; and-   instructions for use.-   Item 36. The kit of Item 35, wherein the background-reducing agent    is linked to a solid phase.-   Item 37. The kit of Item 36, wherein the solid phase is selected    from a coverslip; a particle and a slide.-   Item 38. The kit of Item 35, wherein the background-reducing agent    is in liquid phase.-   Item 39. A background reducing agent, comprising a nucleotide    sequence complementary to a released label of a first probe of the    composition of any one of Items 23-31, and a quench material.-   Item 40. A background reducing agent, comprising a nucleotide    sequence complementary to a released label of a second probe of the    composition of any one of Items 24-31, and a quench material.-   Item 41. A background reducing agent, comprising a nucleotide    sequence complementary to a released activated label of a probe of    the composition of Item any one of Items 25-31, and a quench    material.-   Item 42. A method for detecting a plurality of target molecules, the    method comprising:-   (a) contacting a sample with two or more target-specific binding    partners, wherein each target-specific binding partner comprises a    nucleic acid barcode; and is specific for a different target    molecule;-   (b) contacting the sample with one or more probe-sets wherein each    probe in a probe-set is specific for a different target-specific    binding partner, and wherein each probe-set comprises:-   a first probe, comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a first label; and    -   a cleavage site for a first cleavage agent, wherein the first        cleavage agent is capable of suppressing the first label, and-   a second probe comprising:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a second label;    -   a quench moiety, wherein the quench moiety renders the second        label undetectable; and    -   a cleavage site for the first cleavage agent, wherein the first        cleavage agent is capable of releasing or suppressing the quench        moiety, whereby the second label is rendered detectable;    -   and optionally comprises a cleavage site wherein a second        cleavage agent is capable of releasing or suppressing the second        label;-   (c) detecting signals corresponding to labels of the first probes of    each of the one or more probe-sets;-   (d) contacting the sample with a first cleavage agent, thereby    suppressing the labels of the first probes in each of the one or    more probe-sets; and releasing or suppressing the quench moieties of    the second probes in each of the one or more probe-sets, thereby    activating signals corresponding to the second labels; and-   (e) detecting signals corresponding to the labels of the second    probes of each of the one or more probe-sets.-   Item 43. The method of Item 42, wherein one or more of the    probe-sets further comprises a third probe, comprising:-   (f) in step (b), contacting the sample with a third probe contained    in one or more probe-sets,-   wherein the third probe comprises:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a third label;    -   a quench moiety, wherein the quench moiety renders the third        label undetectable; and    -   a cleavage site for a second cleavage agent, wherein the second        cleavage agent is capable of releasing or suppressing the quench        moiety, whereby the third label is rendered detectable;    -   and optionally comprises a distinct cleavage site wherein a        distinct cleavage agent is capable of releasing or suppressing        the third label of one or more probe-sets; and-   (g) after step (e), contacting the sample with a second cleavage    agent, thereby suppressing the labels of the second probes in each    of the one or more probe-sets; and releasing or suppressing the    quench moieties of the third probes in one or more probe-sets,    thereby activating signals corresponding to the third labels; and-   (h) detecting signals corresponding to the third labels.-   Item 44. The method of Item 43, wherein one or more of the    probe-sets further comprise a subsequent probe, comprising:-   (i) in step (b), contacting the sample with a subsequent probe    contained in one or more probe-set, wherein the subsequent probe    comprises:    -   a nucleic acid sequence complementary to a nucleic acid barcode        of a corresponding target-specific binding partner;    -   a subsequent label; and    -   a quench moiety, wherein the quench moiety renders the        subsequent label undetectable; and    -   a cleavage site, wherein a subsequent cleavage agent is capable        of releasing or suppressing the quench moiety, whereby the        subsequent label is rendered detectable;    -   and optionally comprises a distinct cleavage site wherein a        distinct cleavage agent is capable of suppressing activated        labels from probes in the sample;-   (j) contacting the sample with a subsequent cleavage agent, thereby    suppressing activated labels of the probes in each probe-set; and    releasing the quench moieties of the subsequent probes in each    probe-set, thereby activating signals corresponding to the    subsequent labels; and-   (k) detecting signals corresponding to the subsequent labels; and-   (l) optionally repeating steps (i) through (k).

What is claimed is:
 1. A method for detecting a plurality of targetmolecules, the method comprising: (a) contacting a sample with two ormore target-specific binding partners, wherein each target-specificbinding partner comprises a nucleic acid barcode; and is specific for adifferent target molecule; (b) contacting the sample with one or moreprobe-sets wherein each probe in a probe-set is specific for a differenttarget-specific binding partner, and wherein each probe-set comprises: afirst probe, comprising: a nucleic acid sequence complementary to anucleic acid barcode of a corresponding target-specific binding partner;a first label; and a cleavage site for a first cleavage agent, whereinthe first cleavage agent is capable of releasing the first label, and asecond probe comprising: a nucleic acid sequence complementary to anucleic acid barcode of a corresponding target-specific binding partner;a second label; a quench moiety, wherein the quench moiety renders thesecond label undetectable; and a cleavage site for the first cleavageagent, wherein the first cleavage agent is capable of releasing thequench moiety, whereby the second label is rendered detectable; andoptionally comprises a cleavage site for a second cleavage agent whereinthe second cleavage agent is capable of releasing the second label; (c)detecting signals corresponding to labels of the first probes of each ofthe one or more probe-sets; (d) contacting the sample with a firstcleavage agent, thereby releasing the labels of the first probes in eachof the one or more probe-sets; and releasing the quench moieties of thesecond probes in each of the one or more probe-sets, thereby activatingsignals corresponding to the second labels, and (e) detecting signalscorresponding to the labels of the second probes of each of the one ormore probe-sets.
 2. The method of claim 1, wherein one or more of theprobe-sets further comprises a third probe, and the method furthercomprises: (f) in step (b), contacting the sample with the third probecomprising: a nucleic acid sequence complementary to a nucleic acidbarcode of a corresponding target-specific binding partner; a thirdlabel; and a quench moiety, wherein the quench moiety renders the thirdlabel undetectable; and a cleavage site for the second cleavage agent,wherein the second cleavage agent is capable of releasing the quenchmoiety, whereby the third label is rendered detectable; and optionallycomprises a distinct cleavage site for a distinct cleavage agent capableof releasing the third label; and (g) after step (e), contacting thesample with a second cleavage agent, thereby releasing the labels of thesecond probes in each of the one or more probe-sets; and releasing thequench moieties of the third probes in one or more probe-sets, therebyactivating signals corresponding to the third labels; and (h) detectingsignals corresponding to the labels of the third probes of one or moreprobe sets.
 3. The method of claim 2, wherein one or more of theprobe-sets further comprise a subsequent probe, and the method furthercomprises: (i) in step (b), contacting the sample with a subsequentprobe contained in one or more probe-set, wherein the subsequent probecomprises: a nucleic acid sequence complementary to a nucleic acidbarcode of a corresponding target-specific binding partner; a subsequentlabel; and a quench moiety, wherein the quench moiety renders thesubsequent label undetectable; and a cleavage site for a subsequentcleavage agent, wherein the subsequent cleavage agent is capable ofreleasing the quench moiety, whereby the subsequent label is rendereddetectable; and optionally comprises a distinct cleavage site for adistinct cleavage agent capable of releasing activated labels fromprobes in the sample; (j) contacting the sample with a subsequentcleavage agent, thereby releasing activated labels of the probes in eachprobe-set; and releasing the quench moieties of the subsequent probes ineach probe-set, thereby activating signals corresponding to the labelsof the subsequent probes in each probe-set; and (k) detecting signalscorresponding to the labels of the subsequent probes of each probe-set;and (l) optionally repeating steps (i) through (k).
 4. The method ofclaim 1, wherein the first and second detectable labels of a probe-setare the same.
 5. The method of claim 1, wherein the first and seconddetectable labels of a probe-set are different.
 6. The method of claim2, wherein the two or more of the first, second, and third detectablelabels of a probe-set are the same.
 7. The method of claim 2, whereinthe two or more of the first, second, and third detectable labels of aprobe-set are different.
 8. The method of claim 3, wherein two or moreof the first, second, third, and subsequent labels are the same.
 9. Themethod of claim 3, wherein two or more of the first, second, third, andsubsequent labels are different.
 10. The method of claim 1, furthercomprising washing the sample after contacting the sample with the firstcleavage agent and/or after contacting the sample with the secondcleavage agent.
 11. The method of claim 1, wherein the sample is notwashed after contacting the sample with the first cleavage agent and/orafter contacting the sample with the second cleavage agent.
 12. Themethod of claim 11, wherein the coverslip is not removed.
 13. The methodof claim 1, further comprising increasing the number of nucleic acidbarcodes on a target-specific binding partner, wherein multiple copiesof a corresponding probe bind to multiple copies of the nucleic acidbarcode.
 14. The method of claim 13, wherein the number of nucleic acidbar codes is increased using rolling circle amplification, primerexchange reaction, hybridization chain reaction, or DNA branching. 15.The method of claim 1, wherein the released label of a first probecomprises a nucleotide sequence.
 16. The method of claim 15, furthercomprising contacting the sample with a background-reducing agentcomprising a nucleotide sequence complementary to that of the releasedlabel of the released first probe, wherein binding of thebackground-reducing agent to the released label of the released firstprobe quenches the signal of the label.
 17. A probe-set compositioncomprising: one or more first probes, each comprising: a nucleic acidsequence complementary to a nucleic acid barcode of a correspondingtarget-specific binding partner; a first label; and a cleavage site fora first cleavage agent, wherein the first cleavage agent is capable ofreleasing the first label, and one or more second probes, eachcomprising: a nucleic acid sequence complementary to a nucleic acidbarcode of a corresponding target-specific binding partner; a secondlabel; a quench moiety, wherein the quench moiety renders the secondlabel undetectable; and a cleavage site for the first cleavage agent,wherein the first cleavage agent is capable of releasing the quenchmoiety, whereby the second label is rendered detectable; and optionallycomprises a distinct cleavage site for a distinct cleavage agent capableof releasing the second label.
 18. A kit, comprising: the probe-setcomposition of claim 17; a background-reducing agent; a coverslip; oneor more target-specific binding partners; one or more buffers; one ormore reagents for increasing the number of nucleic acid barcodes of atarget-specific binding partner; one or more cleavage agents; a nuclearcounterstain; and instructions for use.
 19. A background reducing agent,comprising a nucleotide sequence complementary to a released label of afirst probe of the composition of claim 17, and a quench material.
 20. Amethod for detecting a plurality of target molecules, the methodcomprising: (a) contacting a sample with two or more target-specificbinding partners, wherein each target-specific binding partner comprisesa nucleic acid barcode; and is specific for a different target molecule;(b) contacting the sample with one or more probe-sets wherein each probein a probe-set is specific for a different target-specific bindingpartner, and wherein each probe-set comprises: a first probe,comprising: a nucleic acid sequence complementary to a nucleic acidbarcode of a corresponding target-specific binding partner; a firstlabel; and a cleavage site for a first cleavage agent, wherein the firstcleavage agent is capable of suppressing the first label, and a secondprobe comprising: a nucleic acid sequence complementary to a nucleicacid barcode of a corresponding target-specific binding partner; asecond label; a quench moiety, wherein the quench moiety renders thesecond label undetectable; and a cleavage site for the first cleavageagent, wherein the first cleavage agent is capable of releasing orsuppressing the quench moiety, whereby the second label is rendereddetectable; and optionally comprises a cleavage site wherein a secondcleavage agent is capable of releasing or suppressing the second label;(c) detecting signals corresponding to labels of the first probes ofeach of the one or more probe-sets; (d) contacting the sample with afirst cleavage agent, thereby suppressing the labels of the first probesin each of the one or more probe-sets; and releasing or suppressing thequench moieties of the second probes in each of the one or moreprobe-sets, thereby activating signals corresponding to the secondlabels; and (e) detecting signals corresponding to the labels of thesecond probes of each of the one or more probe-sets.